The purpose of these studies was to determine whether lactogen-dependent Nb2-11c cells and lactogen-independent Nb2-SP cells differ with respect to morphology and autocrine growth control. To this end, the ultrastructural and surface morphology of both Nb2 cell lines was analyzed and the autocrine growth modulatory activity of Nb2 cell conditioned medium (Nb2-CM) was determined. The autocrine growth inhibitory activity of Nb2-CM was biologically characterized and attempts were made to biochemically characterize and purify the Nb2 cell autocrine growth inhibitor as well as to determine its mechanism of action. Quantitative analysis of transmission electron micrographs reveals that the ultrastructural morphology of lactogen-dependent Nb2-11c cells differs from that of lactogen-independent Nb2-SP cells. Nb2-11c cells exhibit a greater incidence and volume density of nuclear pockets, whereas the incidence and volume density of lipid droplets is greater in the Nb2-SP cell line. Surface feature of Nb2-11c and Nb2-SP cells, as examined with scanning electron microscopy, and indistinguishable. Nb2-11c and Nb2-SP cells share a common mode of growth control in the form of constitutive secretion of an autocrine inhibitory factor. Medium conditioned by either Nb2-11c or Nb2-SP cells inhibits the growth of both cell lines. Nb2-CM-mediated growth inhibition is dose-dependent and reversible. Nb2-CM does not induce quiescence or cell death, but rather, causes a delay in the progression of cells through all phases of the cell cycle. Nb2 cell proliferation stimulated by a variety of mitogens is inhibited by Nb2-CM. Nb2-CM also has the ability to inhibit the growth of normal rat splenocytes as well as MCF-7 human breast cancer cells. Biochemical analysis of Nb2-CM was equivocal; however, indirect evidence suggests that the autocrine growth inhibitory factor produced by Nb2 cells may be a prostaglandin or another arachadonic acid metabolite since the growth inhibitory activity of Nb2-CM is reduced when CM is prepared in the presence of indomethacin. Interestingly, levels of prostaglandin F₁(α) are elevated in CM-treated culture supernatants. Examination of other signal transduction systems in Nb2 cells suggests that neither cAMP activation, polyamine biosynthesis, nor protein kinase C activation mediate or influence the inhibitory effect of Nb2-CM.

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.

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.

Abstract not available

Lysophosphatidic acid (LPA) is a mitogenic lipid that enhances cell growth, proliferation and motility through binding and activation of at least four receptors, LPA1/Edg2, LPA2/Edg4, LPA3/Edg7, and PPAR and #947;. Here, we show that LPA stimulation inhibits the cell cycle regulator and tumor suppressor, p53. Ten M LPA reduced the cellular levels of total p53 and p53 phosphorylated at serine 15 by approximately 50% in A549 cells and this effect was sustained for at least 6 h. This resulted in a corresponding decrease in p53-mediated transcription. Transient-transfection of the Edg-family LPA receptors, LPA1-3 in HepG2 cells, which do not respond to LPA, also showed this inhibitory response. The response was specific to LPA receptors since neither Gi-coupled M2 muscarinic acetylcholine receptors, nor a mutant LPA1 receptor (LPA1 R124A), which is unable to bind LPA, inhibited p53 activity. Both transient-transfection of the LPA-degrading lipid phosphate phosphatase-1 (LPP-1), or exogenous addition of phospholipase B, which decreases exogenous lysophosphatidate, reversed the LPA receptor-induced decrease in p53-mediated transcription. Although pertussis toxin did not prevent the inhibition of p53, a mutant LPA1 receptor (LPA1 and #8710;361), which lacks the C-terminal PDZ-binding domain, failed to inhibit p53 function. This establishes LPA-mediated inhibition of p53 function requires an interaction with PDZ-containing proteins. These data establish a novel role for LPA-mediated receptor activation in diminishing p53 activity; which, in addition to LPAs well-characterized effects on growth-promoting signaling pathways, is likely to contribute to the survival and proliferation of cancer cells. Of the Edg-family LPA receptors, the LPA1 receptor is the most widely expressed. In the next study, we investigated the agonist-induced endocytosis of the human LPA1 receptor, bearing an N-terminal FLAG epitope tag, in stably transfected HeLa cells. LPA treatment induced the rapid endocytosis of approximately 40% of surface LPA1 within 15 minutes. Internalization was dose dependent and LPA specific since neither lysophophatidylcholine nor sphingosine-1-phosphate induced LPA1 endocytosis. Removing agonist following incubation resulted in LPA1 recycling back to the surface. LPA1 internalization was strongly inhibited by dominant-inhibitory mutants of both dynamin2 (K44A) and Rab5a (S34N). Finally, our results indicate that LPA1 exhibits basal, LPA-dependent internalization in the presence of serum-containing medium.

published_or_final_version / Surgery / Master / Master of Philosophy

A description of two techniques used extensively in this study namely cell counting with a "cell counting plate" and argentation TLC for the separation of ω -6 -fatty acids is given. Zn supplementation into GM of two malignant (BL-6 and Hep- 350) and a non-malignant (LLC-MK) cell line/s resulted in an increased uptake of Zn by the cells and progressively suppressed proliferation of particularly the malignant cells. Zn chelation by EDTA suppressed in vitro proliferation of all 3 cell line, this effect being more pronounced in the malignant cells. A dietary Zn deficiency resulted in alopecia in mice and both a dietary Zn deficiency and Zn excess reduced growth of BL-6 tumours implanted subcutaneously in mice. Zn supplementation into GM progressively increased the uptake of [1-¹⁴C]-LA by BL-6 and LLC-MK cells but had a very slight though irregular effect on this parameter in the Hep- 350 cells. Zn supplementation also stimulated desaturase activity in the BL-6 cells. These results suggested that there are select cell lines whose Δ⁶-desaturase activity responds positively to Zn supplementation (e.g. the BL-6 cells). Delta-6-desaturase activity was also assayed in microsome preparations from different tissues. No enzyme activity was detected in the microsomes prepared from the BL-6 tumours. There was no significant effect with the addition of Zn or EDTA, on Δ⁶-desaturase activity of the regenerating liver microsomes. In the resting liver microsomes this enzyme activity was reduced only when EDTA and Zn were added together and when EDTA was added to the reaction medium as well as to the microsome preparations 2 hr before the enzyme activity assay was initiated. The results of these experiments suggested that the Δ⁶-desaturase enzyme in the microsome preparations may have had an adequate amount of Zn with further additions having no stimulatory effect on the enzyme. Two independent mechanisms of control of cell proliferation by low and high Zn are suggested to operate.

Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.

The glycoprotein stanniocalcin-1 (STC1) is a paracrine factor in mammals which plays roles in various (patho)physiological functions, such as inflammation and carcinogenesis. Considerable numbers of studies showed dysregulation of STC1 expression in different types of human cancers. A previous study from our group, using clinicopathological data of 216 hepatocellular carcinoma (HCC) patients revealed greater STC1 gene expression in tumors than the paired normal samples. However, patient samples with greater STC1 level exhibited smaller tumor size. In fact, multiple cell types, growth factors and matrix components in tumor microenvironment (TME) control cancer progression. Emerging evidence support the important role of infiltrating immune cells on tumor progression. Among those, tumor associated macrophages (TAM) in TME is known to be an essential driver of tumor inflammation and progression, exerting a yin-yang influence to determine if the tumor is suppressed or paving the way to metastasize. Hepatocellular carcinoma (HCC) is mainly caused by chronic inflammation. With hindsight, the roles of STC1 in inflammation and carcinogenesis were documented. However, the observation on the negative correlation of STC1 expression with tumor size in HCC patients and the roles of STC1 on the interactions between tumor cells and macrophages are not clear. In Chapter 2, the inverse correlation of STC1 expression with tumor size was addressed. Human metastatic HCC cell line, MHCC97L which was stably transfected with empty vector (P) and STC1 (S1) were used. Nude mice xenograft model showed that tumor size and volume formed from S1 cells were significantly smaller than that from P cells. The observation agreed with the clinical data aforementioned. In vitro studies demonstrated S1 cells had lower plating efficiency, migratory and proliferative potential, illustrating a lower tumorigenicity. Biochemical analyses on the rate of glycolysis, extracellular O2 consumption, ATP production and Western blot studies on mTOR/p70S6K/rpS6 pathway showed the S1 cells adopted a lower energy metabolism. The data may explain the negative correlation between STC expression level and tumor size. In cancer microenvironment, infiltration of host immune cells, especially macrophages, contributes to inflammation and tumor progression. In Chapter 3, it was hypothesized that cancer cell-derived STC1 alter macrophage functions. Therefore, the effects of STC1-overexpressing MHCC97L on macrophages were studied. To mimic their interactions, Boyden chamber insert model was adopted to co-culture MHCC97L (97L/P and 97L/S1) and THP-1. Our data illustrated 97L/S1 suppressed migratory response of THP-1, with or without the addition of monocyte chemoattractant protein 1 (MCP-1) as the chemoattractant. Quantitative PCR showed downregulation of cytokine/chemokine receptors (CCR2, CCR4, CSF-1R) in THP-1 when co-cultured with 97L/S1. This prompted us to study the alterations of pathways related to cell motility in THP-1 by 97L/S1. Transcriptomic analysis detected 1784 differentially expressed genes (DEGs) between THP-1 cells co-cultured with 97L/P and 97L/S1. Ingenuity Pathway Analysis (IPA) prioritized an inhibition of RhoA signaling, which is known to stimulate cell motility. Western blotting analysis supported the IPA prediction and the cell migration data to show a significant reduction of MLC2 phosphorylation, leading to impaired formation of stress fibers, cell contraction and cell motility. The preceding chapters focused on cancer cell-derived STC1 on HCC cells or THP-1 derived macrophages. In Chapter 4, it was hypothesized that macrophage-derived STC1 may also play a role in macrophage differentiation and inflammation, which modulate tumorigenicity of HCC during macrophage-cancer cell interactions. Thus, the roles of endogenous STC1 in macrophage differentiation and functions were investigated. Using human leukemia monocytic cell line THP-1, a pilot study showed a treatment with phorbol 12-myristate 13-acetate (PMA) significantly upregulated STC1 expression and pro-inflammatory cytokines. In follow-up studies, THP-1 was pharmacologically stimulated to differentiate into (i) classically activated macrophages (CAM)/ M1 state, and (ii) alternatively activated macrophages (AAM)/ M2 state. Greater STC1 expression was found to be associated with CAM. To examine the role of STC1 in CAM, siRNASTC1 was used for gene knockdown. Conditioned medium collected from siRNASTC1-treated CAM inhibited migration of HCC cell line Hep3B. Transcriptomic analysis of siRNASTC1-treated CAM revealed an upregulation on TBC1D3G gene, which is involved in the release of extracellular vesicles (EVs) in macrophage to mediate inflammation. This study demonstrated the association between STC1 and macrophage-mediated inflammation. Collectively, the above studies elucidated the influence of STC1 on cancer cell metabolism, macrophage differentiation and function. It warrants further investigations to unravel the therapeutic potential of STC1 in inflammation and carcinogenesis.