published_or_final_version / Clinical Oncology / Master / Master of Philosophy

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When cells divide, a series of events must proceed in a timely and co-ordinated manner to ensure that all DNA is replicated and partitioned equally between the two daughter cells. A central component of this process is the kinetochore, a large proteinaceous complex (>100 proteins) found within the centromere of all chromosomes. During the dynamic process of cell division, this machinery must be able to capture microtubules, promote chromosome movements towards the spindle midzone and ensure that segregration only occurs once this alignment has been successfully completed. This requires intricate mechanical and regulatory co-ordination between components and it is therefore no surprise that the structures responsible are structurally and functionally varied. It has, however, become clear that many kinetochore proteins assemble into distinct sub-complexes and despite the fact that their specific contributions are well studied, the way the many unique sub-assemblies come together to form a fully operational kinetochore is still poorly understood. Here, chromosome isolation techniques from chicken DT40 cells combined with mass spectrometry employing Stable Isotope Labeling by Amino acids in Cell culture (SILAC), is used to compare the proteome of mitotic chromosomes from different conditional kinetochore knockout (KO) cell lines. This includes components of the inner kinetochore; CENP-C, CENP-T and CENP-W, and a sub-unit of the Ndc80 complex that is important for microtubule attachment. With these large data sets I have focused on the impact these depletions have on the architecture of the holo-kinetochore by measuring the SILAC ratios of individual proteins. From these measurements I can define whether specific components are decreased, increased or unchanged in terms of their abundance on chromosomes in response to the various deletions. I have found that proteins within the same complex typically behave in a similar manner across the different KO conditions. By integrating all of the data sets, dependency networks are revealed, as well as highlighting potential novel kinetochore proteins worthy of further study.

<p>Chronic lymphocytic leukemia (CLL) cells are considered to be derived from antigen-exposed B cells. To further explore the antigen-driven selection behind the leukemogenesis of CLL, we performed immunoglobulin (Ig) specificity screening of 7 CLL cell lines and 23 primary CLL clones from patient peripheral blood. We also included a recombinant monovalent monoclonal antibody (mAb) belonging to a subset of CLL cases with identical or semiidentical heavy chain complementarity determining region 3 (HCDR3) of the IGHV3-21 gene rearrangement. We found CLL mAb specificities against vimentin, filamin B, cofilin-1, proline-rich acidic protein 1, cardiolipin, oxidized low density lipoprotein and Streptococcus pneumoniae polysaccarides. These molecules are functionally associated with microbial infection and/or apoptotic cell removal. An antigen-driven selection would therefore imply that CLL B cell precursors are involved in the elimination and scavenging of pathogens and apoptotic cells, which could trigger the development of the disease.</p><p>The limited in vitro survival of CLL cells makes Epstein-Barr virus (EBV) immortalization of CLL cells a useful experimental model for studies on antibody-specificity screening. Considering the intricate procedure of EBV transformation of CLL cells and the many false cell lines used worldwide, we also wanted to characterize and evaluate the authentic origin of several previously established CLL cell lines and their normal lymphoblastoid counterparts. Three of the CLL cell lines tested were truly authentic (I83-E95, CLL-HG3 and CII), two had features of a biclonal Ig expression (232B4 and WaC3CD5+), one was only tentatively verified (PGA-1), whereas one cell line could not be verified (EHEB) due to lack of original patient cells for comparison. Two of the presumed normal lymphoblastoid cell lines tested were shown to be a neoplastic CLL clone. This study emphasizes the importance of proper cell line authentication and we will continue to verify additional cell lines not yet proven authentic.</p><p>In conclusion, we provide evidence for natural Ab production by CLL cells and suggest that these cells might be derived from B cell precursors involved in the innate immunity and, thus, providing a first-line-defence against pathogens and in elimination of apoptotic cells.</p>

Kisspeptin (encoded by Kiss1) is a hypothalamic neuropeptide that is directly regulated by sex steroids and directly stimulates gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin cell models were established in order to facilitate future molecular analysis of kisspeptin. mHypoA-51 and mHypoA-63 cell lines were found to express kisspeptin, estrogen receptor α and β, substance P, but not tyrosine hydroxyase. Furthermore, estrogen decreased Kiss1 expression in both cell lines. Based on these results, it was concluded that mHypoA-51 and mHypoA-63 are representative of arcuate kisspeptin neurons. Accumulating evidence also indicates that kisspeptin indirectly stimulates GnRH neurons through afferent neurons. Kisspeptin receptor expression was detected in native neuropeptide Y (NPY) neurons. Using the mHypoE-38 cell line, kisspeptin was found to directly regulate NPY mRNA expression and secretion via the ERK1/2 and p38 MAPK pathways. This is the first evidence that kisspeptin directly stimulates NPY neurons to potentially exert indirect effects on GnRH neurons.

Kisspeptin (encoded by Kiss1) is a hypothalamic neuropeptide that is directly regulated by sex steroids and directly stimulates gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin cell models were established in order to facilitate future molecular analysis of kisspeptin. mHypoA-51 and mHypoA-63 cell lines were found to express kisspeptin, estrogen receptor α and β, substance P, but not tyrosine hydroxyase. Furthermore, estrogen decreased Kiss1 expression in both cell lines. Based on these results, it was concluded that mHypoA-51 and mHypoA-63 are representative of arcuate kisspeptin neurons. Accumulating evidence also indicates that kisspeptin indirectly stimulates GnRH neurons through afferent neurons. Kisspeptin receptor expression was detected in native neuropeptide Y (NPY) neurons. Using the mHypoE-38 cell line, kisspeptin was found to directly regulate NPY mRNA expression and secretion via the ERK1/2 and p38 MAPK pathways. This is the first evidence that kisspeptin directly stimulates NPY neurons to potentially exert indirect effects on GnRH neurons.

An intestinal cell line from rainbow trout, Oncorhynchus mykiss, was developed and challenged against several bioactive components. Primary cultures initiated from the distal segment produced the cell line, RTgutGC. RTgutGC showed optimal growth in L15 supplemented with 10-20% fetal bovine serum (FBS) at room temperature. RTgutGC has undergone over 100 passages and stained minimally for β-galactosidase, suggesting this to be an immortal cell line. Late passage cultures gave a consistent polygonal morphology with distinct borders. RTgutGC stained positive for alkaline phosphatase (AP) under certain culture conditions, hence may produce intestinal-specific alkaline phosphatase (IAP). Lipopolysaccharide (LPS) was used as a model microbial endotoxin for determining the sensitivity of the cells to a natural ligand in the gastrointestinal tract (GIT). Exposure of LPS was compared between RTgutGC and two mammalian intestinal cell lines (HT-29 and Caco-2). LPS induced cell death in RTgutGC, potentially through an alternative pathway seen in higher vertebrate response. Cytotoxicity of LPS against RTgutGC, seeded at normal density, was reduced in the presence of glutamine compared to L15 alone (t test, p≤ 0.05). RTgutGC seeded at a super density, where AP was strongly expressed, also showed less toxicity towards LPS. Two isoforms of tumor necrosis factor alpha (TNF-α) transcripts were up-regulated after LPS treatment in RTgutGC. Six rainbow trout cell lines, including RTgutGC, showed constitutive transcript expression of several immune-related genes: Major Histocompatibility (MH) class II α and ß. When MH activity was examined at the protein level, the cell lines showed constitutive expression of MH class I proteins, but not for MH class II molecules. RTS11, a rainbow trout spleen monocyte/ macrophage-like cell line, was the only line to express all MH transcripts and proteins. The utility of the anti-rainbow trout MH protein sera was demonstrated by exposing RTgutGC to poly IC. After a 3 day treatment, RTgutGC showed up-regulation of β2m protein expression. Thus, the cellular and immunological responses in fish intestinal cells can be modeled using the methods presented in this study.

An intestinal cell line from rainbow trout, Oncorhynchus mykiss, was developed and challenged against several bioactive components. Primary cultures initiated from the distal segment produced the cell line, RTgutGC. RTgutGC showed optimal growth in L15 supplemented with 10-20% fetal bovine serum (FBS) at room temperature. RTgutGC has undergone over 100 passages and stained minimally for β-galactosidase, suggesting this to be an immortal cell line. Late passage cultures gave a consistent polygonal morphology with distinct borders. RTgutGC stained positive for alkaline phosphatase (AP) under certain culture conditions, hence may produce intestinal-specific alkaline phosphatase (IAP). Lipopolysaccharide (LPS) was used as a model microbial endotoxin for determining the sensitivity of the cells to a natural ligand in the gastrointestinal tract (GIT). Exposure of LPS was compared between RTgutGC and two mammalian intestinal cell lines (HT-29 and Caco-2). LPS induced cell death in RTgutGC, potentially through an alternative pathway seen in higher vertebrate response. Cytotoxicity of LPS against RTgutGC, seeded at normal density, was reduced in the presence of glutamine compared to L15 alone (t test, p≤ 0.05). RTgutGC seeded at a super density, where AP was strongly expressed, also showed less toxicity towards LPS. Two isoforms of tumor necrosis factor alpha (TNF-α) transcripts were up-regulated after LPS treatment in RTgutGC. Six rainbow trout cell lines, including RTgutGC, showed constitutive transcript expression of several immune-related genes: Major Histocompatibility (MH) class II α and ß. When MH activity was examined at the protein level, the cell lines showed constitutive expression of MH class I proteins, but not for MH class II molecules. RTS11, a rainbow trout spleen monocyte/ macrophage-like cell line, was the only line to express all MH transcripts and proteins. The utility of the anti-rainbow trout MH protein sera was demonstrated by exposing RTgutGC to poly IC. After a 3 day treatment, RTgutGC showed up-regulation of β2m protein expression. Thus, the cellular and immunological responses in fish intestinal cells can be modeled using the methods presented in this study.

Recent experiments are bringing to the fore more and more information about the effects of different treatments on the gene expression of different genes. The results obtained from these experiments show that some definite trends are observed in different genes in the Human Embryonic Kidney and Human Colon Adenocarcinoma Grade II cell lines. The difference in the gene expressions of the two cell lines motivates the problem in this thesis. The thesis provided intervention methods to make the colon cancer cell line genes behave more like their Human Embryonic Kidney cell line counterparts. Two methods of intervention were introduced. The first method was the simpler on-off control intervention while the second method used a more advanced proportional integral control to meet the goal. A comparison of these two intervention methods showed the clear implementational advantages of proportional integral control over on-off control.

Erythropoietin (EPO) is produced in the kidney and in fetal liver in response to hypoxia as well as to CoCl2. The EPO protein and mRNA can be induced in response to both stimuli in the human hepatoma cell (HCC) lines Hep 3B and Hep G2. An oxygen sensing mechanism in which a ligand dependent conformational change in the heme protein produces H2O2 in respone to either hypoxia or Cobalt has been demonstrated. However, an intriguing question can be raised as to why some HCC sublines, such as Hep G2 and Hep 3B are capable of expressing EPO gene, whereas in other HCC sublines, such as J5 and SK-Hep-I are completely devoid of the ability to express EPO gene. Along this line, does ¡§differentiation status¡¨ of these HCC cells play a pivotal role in regulating the expression of EPO gene? Next in line, how a differentiation-associated upregulation of g-glutemylcysteine synthetase (g-GCS), which tightly regulating the biosynthesis of endogenous glutathione(GSH) can modulate the expression of EPO. The objective of this research project was designed to address all these questions. Reported herein are several lines of evidence to demonstrate that endogenous GSH contents do play a pivotal role in the control and regulation of the expression of EPO gene. Firstly, using a group of five HCC lines with varying degrees of differentiation as the experimental model, we demonstrated that the endogenous GSH contents of these HCC cells were differentially upregulated depending on the degree of differentiation with an order of abundance being Hep G2> Hep 3B> J5> Mahlavu> SK-Hep-I. Coincidently, we also found that g-GCS heavy subunit activities as well as its mRNA correlated precisely with this order. Among these HCC cell lines tested, only two well-differentiated sublines, Hep G2 and Hep 3B expressed EPO gene implying that the latter process was dependent upon GSH and suggested a notion that a threshold level might be required for its optimal reactivation. Secondly, to further obtain the evidence to substantiate this possible role of GSH, we then supplemented to the cell culture media with an excessive quantity of nonlethal N-acetylcysteine for the purpose of reinforcing the endogenous GSH biosynthesis. Interestingly, we found that this manipulation could revert the reactivation of EPO gene in cell lines, such as J5 and SK-Hep-I, in which their EPO gene expressions were ortherwise shut down under a normal circumstance. Finally, we were able to demonstrated using RT-PCR and western blotting that the expression of EPO gene was reverted in GCS30, a SK-Hep-I subline that was permanently transfected with g-GCSh and is capable of overly expressing endogenous GSH. Taken together, we demonstrated herein for the first time that, besides hypoxia and CoCl2, endogenous GSH contents can also act as a positive regulator for the expression of EPO gene. The underlying mechanism of how GSH exerts its action in the regulation of EPO expression awaits further clarification.