Cold-Temperature Adaptation of Muscle Creatine Kinase from an Antartic Teleost (\u3cem\u3eChaenocephalus Aceratus\u3c/em\u3e)

The white muscle of Chuenocephulus aceratus, an Antarctic teleost of the Channicthyidae family, has a compromised glycoiytic capacity and this fish cannot depend on glycolysis for rapid ATP generation For C. aceratus, creatine kinase (CK) and phosphocreatine (PCr) reserves comprise the metabolic pathway that may supplement and overcome this deficiency in energy transduction. Two conditions, low glycolytic capacity and evolution in a chronically cold habitat (-1.86°C), give us reason to believe that C. aceratus muscle CK (MMCK) has been subjected to strong selective pressure. Thus, the hypothesis of this thesis is that MMCK fiom C. aceratus white muscle exhibits a high specific activity. In order to test this hypothesis, MMCK from C. aceratus white (glycolytic) muscle was purified. This revealed that C. aceratus expresses two cytosolic isoforms of MMCK. Mammals, birds, and most fish express only one MMCK. Thermal stability studies indicate that C. aceratus MMCKs exhibit thermal denaturation after a 30 minute incubation at temperatures greater-than 10°C. At O°C, the average apparent Vmax was found to be 3-to-5 times higher than that predicted fiom a Q10 analysis. The estimate of activation enthalpy for C. aceratus MMCK was 7 kcal/mol lower than that of rabbit MMCK and the activation free energy estimate of C. aceratus MMCK was 1 kcal/mol lower. This helps explain C. aceratus MMCK\u27s high catalytic activity at low temperature, which was found to be greater-than eurythermal fish MMCK activities at 25°C. Kinetic constant estimates indicate that C. aceratus MMCKs have an affinity for ADP (Km= 0.06 mM) and PCr (Km= 17 mM) that is similar to what has been reported for other MMCKs. All C. aceratus CK cDNAs were cloned and sequenced, which confirmed that C. aceratus expresses two distinct isoforms of MCK. The tissues that express muscle, brain, and mitochondria1 CK mRNAs were determined. This is a characteristic of each CK isoform and C. aceratus MCK mRNAs were obsevered only in skeletal muscle. The evidence presented in this thesis demonstrates that one or both MMCKs have undergone cold-temperature adaptation

Neoplastic transformation of breast epithelial cells by genotoxic stress

<p>Abstract</p> <p>Background</p> <p>Exposure to genotoxic stresses such as radiation and tobacco smoke can cause increased cancer incidence rate as reflected in an in depth meta-analysis of data for women and breast cancer incidence. Published reports have indicated that exposures to low dose radiation and tobacco smoke are factors that contribute to the development of breast cancer. However, there is a scarcity of information on the combinatorial effects of low dose radiation and tobacco smoke on formation and progression of breast cancer. The combination of these two genotoxic insults can induce significant damage to the genetic material of the cells resulting in neoplastic transformation.</p> <p>Methods</p> <p>To study the effects of low dose ionizing radiation and tobacco smoke on breast cells, MCF 10A cells were treated either with radiation (Rad - 0.1 Gray) or cigarette smoke condensate (Csc - 10 microgram/ml of medium) or a combination of Rad + Csc. Following treatments, cells were analyzed for cell cycle distribution patterns and the ability to extrude the Hoechst 33342 dye. In addition, <it>in vitro </it>invasion and migration as well as mammosphere formation assays were performed. Finally, differential gene expression profiles were generated from the individual and combination treatment.</p> <p>Results</p> <p>Exposure of MCF 10A cells to the combination of radiation plus cigarette smoke condensate generated a neoplastic phenotype. The transformed phenotype promoted increased mammosphere numbers, altered cell cycle phases with a doubling of the population in S phase, and increased invasion and motility. Also, exclusion of Hoechst 33342 dye, a surrogate marker for increased ABC transporters, was observed, which indicates a possible increase in drug resistance. In addition, changes in gene expression include the up regulation of genes encoding proteins involved in metabolic pathways and inflammation.</p> <p>Conclusions</p> <p>The results indicate that when normal breast cells are exposed to low dose radiation in combination with cigarette smoke condensate a phenotype is generated that exhibits traits indicative of neoplastic transformation. More importantly, this is the first study to provide a new insight into a possible etiology for breast cancer formation in individuals exposed to low dose radiation and tobacco smoke.</p

Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing

Organ-specific isogenic metastatic breast cancer cell lines exhibit distinct Raman spectral signatures and metabolomes

Molecular characterization of organ-specific metastatic lesions, which distinguish them from the primary tumor, will provide a better understanding of tissue specific adaptations that regulate metastatic progression. Using an orthotopic xenograft model, we have isolated isogenic metastatic human breast cancer cell lines directly from organ explants that are phenotypically distinct from the primary tumor cell line. Label-free Raman spectroscopy was used and informative spectral bands were ascertained as differentiators of organ-specific metastases as opposed to the presence of a single universal marker. Decision algorithms derived from the Raman spectra unambiguously identified these isogenic cell lines as unique biological entities – a finding reinforced through metabolomic analyses that indicated tissue of origin metabolite distinctions between the cell lines. Notably, complementarity of the metabolomics and Raman datasets was found. Our findings provide evidence that metastatic spread generates tissue-specific adaptations at the molecular level within cancer cells, which can be differentiated with Raman spectroscopy.National Institute of Biomedical Imaging and Bioengineering (U.S.) (9P41EB015871-26A1

Localized Hypoxia Results in Spatially Heterogeneous Metabolic Signatures in Breast Tumor Models

Tumor hypoxia triggers signaling cascades that significantly affect biologic outcomes such as resistance to radiotherapy and chemotherapy in breast cancer. Hypoxic regions in solid tumor are spatially heterogeneous. Therefore, delineating the origin and extent of hypoxia in tumors is critical. In this study, we have investigated the effect of hypoxia on different metabolic pathways, such as lipid and choline metabolism, in a human breast cancer model. Human MDA-MB-231 breast cancer cells and tumors, which were genetically engineered to express red fluorescent tdTomato protein under hypoxic conditions, were used to investigate hypoxia. Our data were obtained with a novel three-dimensional multimodal molecular imaging platform that combines magnetic resonance (MR) imaging, MR spectroscopic imaging (MRSI), and optical imaging of hypoxia and necrosis. A higher concentration of noninvasively detected total choline-containing metabolites (tCho) and lipid CH3 localized in the tdTomato-fluorescing hypoxic regions indicated that hypoxia can upregulate tCho and lipid CH3 levels in this breast tumor model. The increase in tCho under hypoxia was primarily due to elevated phosphocholine levels as shown by in vitro MR spectroscopy. Elevated lipid CH3 levels detected under hypoxia were caused by an increase in mobile MR-detectable lipid droplets, as demonstrated by Nile Red staining. Our findings demonstrate that noninvasive MRSI can help delineate hypoxic regions in solid tumors by means of detecting the metabolic outcome of tumor hypoxia, which is characterized by elevated tCho and lipid CH3

Peptides Derived from Type IV Collagen, CXC Chemokines, and Thrombospondin-1 Domain-Containing Proteins Inhibit Neovascularization and Suppress Tumor Growth in MDA-MB-231 Breast Cancer Xenografts

Angiogenesis or neovascularization, the process of new blood vessel formation from preexisting microvasculature, involves interactions among several cell types including parenchymal, endothelial cells, and immune cells. The formation of new vessels is tightly regulated by a balance between endogenous proangiogenic and antiangiogenic factors to maintain homeostasis in tissue; tumor progression and metastasis in breast cancer have been shown to be angiogenesis-dependent. We previously introduced a systematic methodology to identify putative endogenous antiangiogenic peptides and validated these predictions in vitro in human umbilical vein endothelial cell proliferation and migration assays. These peptides are derived from several protein families including type IV collagen, CXC chemokines, and thrombospondin-1 domain-containing proteins. On the basis of the results from the in vitro screening, we have evaluated the ability of one peptide selected from each family named pentastatin-1, chemokinostatin-1, and properdistatin, respectively, to suppress angiogenesis in an MDA-MB-231 human breast cancer orthotopic xenograft model in severe combined immunodeficient mice. Peptides were administered intraperitoneally once per day. We have demonstrated significant suppression of tumor growth in vivo and subsequent reductions in microvascular density, indicating the potential of these peptides as therapeutic agents for breast cancer

Effect of hypoxic conditions on the expression of the DDX3 gene in breast epithelial cells.

<p>MCF 10A (a & c) and MCF 7 (b & d) cells were cultured under normoxic conditions (20% O₂) or subjected to 200 µM CoCl₂ or 1% O₂ for 8, 12 and 24 h (a & b) qRT-PCR analysis was performed using specific primers for human DDX3 and HPRT as an internal normalization control. The expression level under normoxia was set to 1. DDX3 protein levels (c & d) in cell lysates of MCF 10A and MCF 7 cells were determined by immunoblots using anti-DDX3 antibodies. In these cases, HIF-1α and GAPDH served as controls indicating hypoxic conditions and equal protein loading respectively. Error bars represent ±SD.</p

ChIP assay: <i>in vivo</i> binding of HIF-1 to the DDX3 promoter in MCF 10A cells.

<p>At the top of the gel is a schematic representation of the DDX3 promoter. Arrows flank the region (-423 to -115) amplified by PCR with DDX3 promoter specific primers. Gel shows: lane 1- molecular weight (MW) marker, lane 2- total input chromatin, lane 3-acetyl histone H3 precipitation, lane 4-anti-HIF-1α precipitation under hypoxic conditions, lane 5-anti-HIF-1α under normoxic conditions, and lane 6-anti-actin precipitation. Identical volumes from each final precipitation were used for PCR (except for the input chromatin, which was diluted 100x).</p

Specificity of HIF-1α dependent activation of the DDX3 gene.

<p>Stable MCF 10A and MCF 7-HIF-1α shRNA clones were generated. (a) MCF 10A-shHIF-1α and (b) MCF 7-shHIF-1α cells were subjected to 200 µM CoCl₂ for the indicated times. qRT-PCR analysis was performed using specific primers for human DDX3, HIF-1α and HIF-2α. DDX3, HIF-1α, and HIF-2α protein levels in cell lysates of MCF 10A-shHIF-1α (c) and MCF 7-shHIF-1α (d) cells were examined by immunoblots using protein specific antibodies. GAPDH served as a loading control. Error bars represent ±SD.</p

Correlation between HIF-1α and DDX3 protein expression in MDA-MB-231 breast cancer cells.

<p>(a) Immunoblot showing expression levels of HIF-1α following CoCl₂ treatment in HIF-1α knockdown cells. (b) Histogram showing relative fold expression of DDX3 mRNA protein levels of MDA-MB-231 versus MDA-MB-231-shHIF-1α cells. Expression of DDX3 was downregulated in MDA-MB-231-shHIF-1α relative to MDA-MB-231 cells at both the mRNA and protein levels. (c) Immunohistochemical staining for HIF-1α and DDX3 in sequential slices of a MDA-MB-231 tumor xenograft. Tumor sections were stained with antibodies that are specific for HIF-1α and DDX3. Samples were counterstained with hematoxylin to reveal morphology. Error bars represent ±SD.</p