One of the main results of Rb-E2F pathway disruption is deregulation of the E2F family of transcription factors, which can lead to inappropriate proliferation, oncogenic transformation, or the induction of apoptosis. Given the potential negative biological effects associated with deregulated E2F activity, it is of great importance to study E2F targets that mediate these effects. In Part I of this manuscript, we identify the RhoBTB2 putative tumor suppressor gene as a direct physiological target of the E2F1 transcription factor. We find that RhoBTB2 is highly upregulated during mitosis due in part to E2F1, and that overexpression of RhoBTB2 increases the S-phase fraction and slows the rate of proliferation. We also find RhoBTB2 similarly upregulated during drug-induced apoptosis due primarily to E2F1 and that knockdown of RhoBTB2 expression via siRNA slows drug-induced apoptosis. Taken together, we describe RhoBTB2 as a novel direct target of E2F1 with roles in cell cycle and apoptosis. In Part II, we independently identify from cancer cell lines two novel variants from the promoter of E2F1 target MCL-1---MCL-1 +6 and +18---as initially published by Moshynska et al (1). In contrast to Moshynska et al., we find the variant promoters identically present in both cancerous and adjacent noncancerous clinical lung samples, suggesting that the variants are germ-line encoded. We also find the variant promoters prevalent in genomic DNA derived from healthy control samples and present at frequencies similar to that observed in cancerous cell lines. In further contrast, we find the activity of the MCL-1 +6 and +18 promoters approximately 50% less than the common MCL-1 +0 promoter---both during normal cellular homeostasis and under conditions that actively induce Mcl-1 transcription. Given our results and those of others, we conclude that the MCL-1 +6 and +18 promoters are likely benign polymorphisms and do no [sic] represent a reliable prognostic marker for CLL as reported by Moshynska et al.

Inheritance of gene expression patterns through multiple rounds of cell division is crucial for the normal development of multi-cellular organisms and is mediated by epigenetic mechanisms. Many epigenetic mechanisms are believed to involve heritable changes to chromatin structure. This includes maintenance of transcriptional repression by Polycomb Group (PcG) proteins. It is unknown how PcG-dependent repression is maintained during or re-established after mitosis, a process that involves many physical and biochemical changes to chromatin. Understanding the behavior of PcG proteins during mitosis is key to answering this question: if PcG proteins remain bound in mitosis they may constitute the memory themselves, or else transcriptional memory must reside elsewhere, such as in the altered chromatin structures induced by PcG proteins. PcG protein association with chromosomes in mitosis in Drosophila S2 cells was examined by immunofluorescence and cellular fractionation. PcG proteins are associated with mitotic chromosomes, which is consistent with a role in carrying information about transcriptional repression through mitosis. Localization of PcG proteins to specific sites in the genome was assessed by chromatin immunoprecipitation (ChIP) followed by genome-wide sequencing (ChIP- SEQ) on mitotic cells. A method for isolating pure populations of mitotic cells was developed to access PcG protein localization in mitosis unambiguously. PcG proteins were not detected at well-characterized PcG targets including Hox genes on mitotic chromosomes, but a covalent modification of histone H3 associated with PcG- dependent repression, trimethylation of lysine 27 (H3K27me3), is retained at these sites. Two PcG proteins Posterior Sex Combs (PSC) and Polyhomeotic (PH) remain at about 10% of their interphase binding sites in mitosis. PSC and PH are preferentially retained in mitosis at sites that overlap recently described borders of chromatin domains (1), including sites that overlap domain borders flanking Hox gene clusters. These persistent binding sites may serve to nucleate re-establishment of PcG binding at target genes upon mitotic exit, perhaps with assistance of H3K27me3. Thus PcG proteins may form part of the transcriptional memory carried through mitosis, but perhaps not by persistent association at the targets of repression. Retention of elements at chromatin boundaries in mitosis may serve as a general mechanism for epigenetic memory.

No description available.

No description available.

The cancer antigen 125 (CA125) is a cell-surface mucin which is over-expressed by the majority of ovarian cancers. However, its biology and the role it plays in ovarian cancer is largely unknown, although other cell-surface mucins have been shown to play a role in apoptosis, cell growth and tumour immune evasion. To analyse the function of CA125 in ovarian cancer, we initially knocked down the expression of CA125 using RNA interference. Knocking down CA125 expression using in vitro transcribed short interfering RNAs (siRNAs) induced a potent cell death response, which has been well characterised in the literature as an induction of an interferon response and resulting in cell apoptosis. Subsequently, using the short hairpin RNA expression vector, pSUPER, which has been shown to knock down genes with high efficiency with reduced off-target affects, we generated stable sub-lines of the ovarian cancer cell line, OVCAR-3, which had been transfected with pSUPER constructs targeting CA125. Intriguingly, these sub-lines had a range of abnormal mitotic events and nuclear defects. However, there was no clear association with the level of CA125 knock down. This could be either due to clonal selection from the parent OVCAR-3 cell line or in addition to CA125 knock down, additional genetic changes are required to occur to favour a state of survival. Similar to the in vitro data, xenografts of the sub-clones into SCID mice generated inconclusive results as to whether CA125 knock down contributes to tumour growth, invasion and metastasis in vivo. More recently, we have been able to achieve high levels of short-term CA125 knock down using synthetic siRNAs designed to reduce off-target affects. These preliminary in vitro and in vivo experiments conducted with pSUPER sub-lines should be repeated using synthetic siRNAs to confirm the role of CA125 in this context. Given the role which the cytoplasmic tail of cell-surface mucins plays in its function, we generated a polyclonal antibody recognising the CA125 cytoplasmic tail, designated M16.1. Immunofluorescence imaging of CA125 in ovarian cancer cell lines, OVCAR-3 and PEO-1, using the OC125 extracellular domain antibody indicated cell-surface localisaton of CA125. However, in addition to the cell-surface localisation, the M16.1 antibody localised to the cell cytoplasm, indicating cleavage and release of the CA125 cytoplasmic tail into the cytosol. Additionally, M16.1 co-localised with α-tubulin at perinuclear sites and to areas resembling microtubule organising centres. However, M16.1 did not co-localise with γ-tubulin at the centrosome, indicating association with non-centrosomal microtubules. Furthermore, depolymerisation of microtubules on ice for 1 hour resulted in loss of diffuse cytoplasmic M16.1 staining but co-localisation between M16.1 and α-tubulin at non-centrosomal sites remained. Intriguingly, when microtubules were allowed to reform at 37oC in PEO-1 cells which had CA125 knocked down by synthetic siRNAs, the ability to reform radial asters was impaired, possibly indicating the CA125 cytoplasmic tail involvement in anchoring microtubules to non-centrosomal sites. Furthermore, we also cloned a portion of CA125 encompassing the cytoplasmic tail, transmembrane domain and 9 tandem repeats. When this construct was transfected into COS-1 cells, the CA125 cytoplasmic tail localised to microtubule bundles during metaphase. Mitotic involvement of the endogenous CA125 cytoplasmic tail was confrmed in OVCAR-3 and PEO-1 cells using M16.1. Given this association and also the results from the pSUPER sub-lines with CA125 knockdown, CA125 may be involved in controlling the fidelity of mitosis, which is grossly altered during tumourigenesis. More recently, it was identified that galectin-1 (Gal-1), an S-type lectin, is a ligand for CA125. Gal-1 is a potent inducer of T cell apoptosis and has been implicated as playing a major role in immune evasion for cancer cells. Consequently, we analysed the expression of CA125 and Gal-1 in ovarian cancer and confirmed the two molecules were expressed concurrently at the mRNA level by RT-PCR. Moreover, immunofluorescence studies also confirmed that CA125 and Gal-1 interacted with each other at the cell-surface of 27/87 cells, an ovarian cancer cell-line. Therefore, we hypothesised that CA125 presents Gal-1 to the immune system, which then induces T cell apoptosis and allows the tumour to escape the immune system. However, CA125 did not protect tumour cells from recognition or killing by T cells, which was shown by no differences in IFN-γ secretion or tumour lysis by cytotoxic T cells using influenza peptide pulsed pSUPER sub-lines with CA125 knockdown. The work described in this thesis suggests that CA125 plays a major role in the aetiology and progression of ovarian cancer through its actions on mitosis, microtubule organisation and immune evasion.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph.D.) - University of Glasgow, 2008. / Ph.D. thesis submitted to the Division of Biochemistry and Molecular Biology, Biomedical and Life Sciences (IBLS), University of Glasgow, 2008. Includes bibliographical references. Print version also available.

Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, May 2005. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Environmental Health Sciences. Includes bibliographical references. Available online via OhioLINK's ETD Center.