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    N-Methyl-D-Aspartate Receptor Hypofunction in Meg-01 Cells Reveals a Role for Intracellular Calcium Homeostasis in Balancing Megakaryocytic-Erythroid Differentiation
    (Thieme Gruppe, 2020-04-14) Hearn JI; Green TN; Chopra M; Nursalim YNS; Ladvanszky L; Knowlton N; Blenkiron C; Poulsen RC; Singleton DC; Bohlander SK; Kalev-Zylinska ML
    The release of calcium ions (Ca2+) from the endoplasmic reticulum (ER) and related store-operated calcium entry (SOCE) regulate maturation of normal megakaryocytes. The N-methyl-D-aspartate (NMDA) receptor (NMDAR) provides an additional mechanism for Ca2+ influx in megakaryocytic cells, but its role remains unclear. We created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene, which encodes an obligate, GluN1 subunit of the NMDAR. We found that compared with unmodified Meg-01 cells, Meg-01-GRIN1 -/- cells underwent atypical differentiation biased toward erythropoiesis, associated with increased basal ER stress and cell death. Resting cytoplasmic Ca2+ levels were higher in Meg-01-GRIN1 -/- cells, but ER Ca2+ release and SOCE were lower after activation. Lysosome-related organelles accumulated including immature dense granules that may have contributed an alternative source of intracellular Ca2+. Microarray analysis revealed that Meg-01-GRIN1 -/- cells had deregulated expression of transcripts involved in Ca2+ metabolism, together with a shift in the pattern of hematopoietic transcription factors toward erythropoiesis. In keeping with the observed pro-cell death phenotype induced by GRIN1 deletion, memantine (NMDAR inhibitor) increased cytotoxic effects of cytarabine in unmodified Meg-01 cells. In conclusion, NMDARs comprise an integral component of the Ca2+ regulatory network in Meg-01 cells that help balance ER stress and megakaryocytic-erythroid differentiation. We also provide the first evidence that megakaryocytic NMDARs regulate biogenesis of lysosome-related organelles, including dense granules. Our results argue that intracellular Ca2+ homeostasis may be more important for normal megakaryocytic and erythroid differentiation than currently recognized; thus, modulation may offer therapeutic opportunities.
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    Interactions between MCF-7 and MDA-MB-231 breast cancer cell lines and neutrophils : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Anatomy and Physiology at Massey University, Albany, New Zealand
    (Massey University, 2019) Holland, Sherina
    The tumour micro-environment (TME) has an essential role in tumour development and progression. Immune cells recruited to the site of the tumour secrete soluble factors such as proteinases, growth factors, survival factors and angiogenic factors into the TME. The secretion of these factors is up-regulated via inflammatory mediators secreted by tumour cells, resulting in a pro-malignant cycle between the cancer and immune cells. A greater understanding of the molecular mechanisms underpinning these interactions is required, as this will assist towards identifying potential new drug targets for cancer and ultimately, will aid in the long-term development of targeted and effective treatments for breast cancer and MBC. The activities of certain immune cells, such as tumour associated macrophages, have been reasonably well characterised in cancer, however, until recently, less was known regarding the role of neutrophils in tumour progression. The goal of the research described in this thesis was to determine whether soluble factors secreted by breast cancer cells might alter the phenotype or lifespan of neutrophils. The latter may allow neutrophils sufficient time to participate in activities within the TME that may either help or hinder tumour progression, while soluble factors released by the neutrophils might influence the invasiveness of breast cancer cells. To investigate whether soluble factors released by breast cancer cells could delay neutrophil apoptosis, neutrophils were cultured in conditioned medium (CM) prepared from highly metastatic MDA-MB-231 or poorly metastatic MCF-7 cells. Flow cytometry experiments showed a delay in apoptosis for neutrophils cultured in MDA-MB-231 CM, but not MCF-7 CM. Quantitative RT-PCR was used to measure neutrophil mRNA expression of pro- versus anti-apoptosis peptides; neutrophils incubated in MDA-MB-231 CM, but not MCF-7 CM, demonstrated a significantly higher expression of the anti-apoptosis peptide BCL2 (A1) and significantly lower expression of the pro-apoptosis peptide BAK compared to control. Western blots showed extensive caspase-8 activation for neutrophils cultured in MCF-7 CM, consistent with apoptosis, whilst neutrophils cultured in MDA-MB-231 CM showed little activation of caspase-8, indicating low levels of apoptosis. The soluble factor contained within the MDA-MB-231 CM, responsible for the delay in neutrophil apoptosis was found to be heat stable and have a molecular weight of between 10-100kDA. Prostaglandin E2 (PGE2) was identified as a potential candidate molecule, as it is a heat stable lipid, and when bound to plasma proteins, fits the molecular weight criteria. In addition, neutrophils cultured with 10μM native or heat treated PGE2 demonstrated a delay in apoptosis, however, this was to a lesser extent compared to neutrophils cultured in MDA-MB-231 CM. Cycoloxygenase-2 (COX-2), the enzyme responsible for PGE2 synthesis, was shown to be expressed in MDA-MB-231 cells but not MCF-7 cells, which is in agreement with the results demonstrating a delay in apoptosis for neutrophils cultured in MDA-MB-231 CM but not MCF-7 CM. Freshly isolated human neutrophils, obtained from the peripheral blood of healthy volunteers, cultured in MDA-MB-231 or MCF-7 CM for 7hrs were not polarised toward a pro or anti-tumour phenotype, as determined via the expression of ICAM-1 and MMP-9. Finally, to investigate whether neutrophils could influence the process of EMT and alter the migration of breast cancer cells, neutrophils were indirectly cultured, via transwell plates, with MDA-MB-231 or MCF-7 cells. Neutrophils were not found to enhance the migration of the cancer cells, as determined via a wound scratch assay. Likewise, neutrophils were not shown to influence the process of EMT in the cancer cells, as determined by changes to cell morphology or the expression of EMT markers.
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    In vitro systems to study the relationship between apoptosis in multicellular organisms and yeast : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Turitea, Palmerston North
    (Massey University, 2000) Haywood, Annika Fleur Margo
    Apoptosis is a distinct form of cell death that is characterised by specific morphological and biochemical markers, such as chromatin condensation and internucleosomal DNA cleavage. This type of cell death is evolutionarily conserved in higher eukaryotes. Homologues of the main apoptosis regulators, such as the Bcl-2 family of proteins and caspases, have been found in multicellular organisms. However, homologues of these proteins have not been found in the unicellular organism Saccharomyces cerevisiae, although in certain circumstances S. cerevisiae will exhibit features of apoptosis. In this project, we developed in vitro systems to explore the relationship between mammalian apoptosis and any similar mechanism that may be present in yeast. Components derived from yeast and mammalian cells were incubated together in vitro and assessed for the activation of apoptosis. Rat cytochrome c activates apoptosis in mammalian cell-free extracts (human neuroblastoma SY5Y cells). Internucleosomal DNA cleavage was observed in S. cerevisiae spheroplasts when they were incubated in mammalian cell-free extracts activated by rat cytochrome c. Although yeast cytochrome c is similar to rat cytochrome c, it failed to induce apoptosis in mammalian cell-free extracts. Yeast cytosol caused internucleosomal DNA cleavage in PCl2 nuclei. This cleavage was enhanced by rat cytochrome c and was mostly inhibited by the caspase inhibitor DEVD-CHO, but only in the presence of rat cytochrome c. Yeast cytosol did not cause chromatin condensation in PCl2 nuclei or cleavage of Parp (a downstream caspase substrate). Yeast cytosol was therefore unable to induce apoptosis in PCl2 nuclei. Mitochondria play a central role in most forms of mammalian apoptosis. We developed a cell-free system in which we could examine the role of mitochondria in apoptosis. We attempted to activate apoptosis in SY5Y cytosol by the addition of mitochondria subjected to rupture-inducing treatment, with limited success. However, we found that mitochondria purified from healthy SY5Y cells protected PCl2 nuclei from undergoing apoptosis in vitro.
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    Structural characterization of the SAC domain of the Par-4 protein by NMR spectroscopy : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Physics at Massey University, Turitea, Palmerston North, New Zealand
    (Massey University, 2010) Kate, Sachin Raghunath
    Prostate Apoptosis Response-4 protein (Par-4): Par-4 gene, first identified in prostate cancer cells undergoing apoptosis, encodes a pro-apoptotic protein. Par-4 selectively induces apoptosis in cancer cells causing regression of tumors in animal models. Par-4 induces apoptosis in androgen independent prostate cancer cells and Rastransformed cells but not in androgen-dependent normal cells. Apoptosis induction by the Par-4 involves a complex mechanism that requires activation of the Fas death receptor signalling pathway and coparallel inhibition of NF-kB transcription activity. Par-4 expression is elevated in various neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's diseases and stroke. Rat Par-4 MW 36kDa, full length aa332 protein that shows high homology to human Par-4. Par-4 has two putative nuclear localization sequences NLS1 (aa20-25) and NLS2 (aa 137-153) localized to the N-terminal half of the molecule. SAC domain: Selectivity for apoptosis induction in cancer cells 59-aa (aa137-195) is necessary for apoptosis. Leucine zipper(LZ) domain of 41-aa (aa 292-332) at C terminus which binds to zinc finger domain of aPKC isoforms, Wilms tumor(WT1) protein, p62 and Dlk. Par-4 Δleucine zip aa1-265, zipperless domain. Different types of expression vectors have been tried for expression of the Par-4 SAC domain in E. coli cells. The first expression vector was H-MBP-3C was tried with E.coli rosetta cells. The fusion protein expressed with this vector was not stable. The Par-4 SAC domain protein gene sequence was cloned in different trial vectors. In the beginning we decided to use a vector without any fusion tag, the pPROEX-HTb which shows low level of expression in E.coli BL21 (DE3) CP cells. The pETTEV expression vector with a thioredoxin tag shows good expression level and fusion protein stability. The protein sample after purification was characterized by proton NMR spectroscopy, CD and DLS which does not show any presence of protein folding. pH titration by using NMR spectroscopy to simultaneously observe the protonation state of different ionizing functional groups in peptides and proteins with both acidic and basic condition was also done. There was no evidence of any protein folding in the acidic as well as basic pH range. This suggests that protein is natively unfolded.
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    Regulation of tight junction proteins during engorgement of the mammary gland : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2006) Cooper Phyn, Claire Vanessa
    Extended periods of milk accumulation result in loss of secretory activity, increased apoptosis and eventually, involution of mammary glands. This process is associated with increased permeability of the tight junction (TJ) complexes between adjacent mammary epithelial cells (MECs). The change in cell shape during mammary engorgement from a cuboidal to a flattened morphology may initiate changes in protein and gene expression (mechanotransduction) that trigger these processes. Therefore, this study examined the regulation of the major TJ protein components during mammary engorgement, and in particular the role of physical distension of the mammary epithelium in the regulatory process. Expression of the integral transmembrane TJ proteins, occludin and claudin-1, and the cytoplasmic TJ protein, ZO-1, were down-regulated in both bovine and rat mammary glands during the early stages of mammary apoptosis and involution following the abrupt cessation of milk removal. In the rat, these responses were locally regulated as they occurred only in teat-sealed glands in a hemi-suckled model. Furthermore, the down-regulation of TJ proteins is consistent with a loss of TJ integrity during mammary engorgement. Induced physical distension of rat mammary glands in vivo transiently up-regulated the expression levels of occludin protein and mRNA, and ZO-1 mRNA, followed by an accelerated decrease in expression compared with the effects of milk accumulation alone. This was associated with the initiation of apoptosis, the up-regulation of the pro-apoptotic factor pSTAT3, and the down-regulation of the cell-ECM survival factor βl-integrin. An in vitro model was also developed to stretch MECs, mimicking the flattening in cell shape during mammary engorgement in vivo. While stretching MECs in vitro did not conclusively alter TJ protein expression, the overall results of this project support further investigation into the role of the TJ complex in mechanotransduction pathways. In addition, the results point to crosstalk between cell-ECM survival signalling and STAT3 death signalling as a candidate for regulation by physical distension of the mammary epithelium. In conclusion, this study supports the hypothesis that physical distension during engorgement of the mammary glands with milk is a primary trigger initiating apoptosis of MECs through changes in the regulation of gene pathways controlling cell survival and death, and the disruption of TJ function.