The present work is to understand how cells communicate with each other and with other cells and the abnormalities in Quorum Sensing (QS) that permit cancer stem/progenitor (S/P) cells to far exceed normal homeostatic cell densities. In the present study a p190BCR-ABL driven pre-B acute lymphoblastic leukemia (ALL3) cell line derived from the pleural fluid of a terminally ill patient with acute lymphoblastic leukemia (ALL) was used to test the QS hypothesis in leukemia. Our findings suggest that the Ph+ ALL population achieves dominance by functioning as a collective aberrant ecosystem subject to defective QS regulatory mechanisms. Chapter 1 provides brief introduction of quorum sensing behavior in the bacteria and also in invertebrate system. It also describe the leukemia.
In Chapter 2 the motivation is to understand the collective decision making by leukemic cancer cells. We found that ALL3 cells don’t grow at low density (LD) in liquid media but grow progressively faster at increasingly high cell densities (HD) in contrast to other long-established leukemic cell lines that grow well at very low starting cell densities. The supernates collected from ALL3 cells grown at high starting cell densities, normal primary S/P cells (cord blood cells), CML leukemic cell lines, and leukemic patient cells stimulated the growth of the LD ALL3 cells. I performed various cell growth assays, single cell studies, proliferation assays, and apoptosis assays to understand the behavior of ALL3 cells cultured at different cell densities. I also used microarray analysis of the LD ALL3 cells after stimulation with ALL3 HDSN to identify signaling networks involved when the non-growing LD ALL3 cells are stimulated. The importances of our findings are discussed in the Chapter 2 in detail.
In Chapter 3, I found that exosomes can also stimulate the growth of the LD ALL3 cells. Exsomes are nanometer size vesicle released by cells and they contain various biomolecules like proteins, RNAs, DNAs, and lipids within them. They have an important role in cell-cell communications. I found that the non-growing LD ALL3 cells could be stimulated to grow in the presence of exosomes released by ALL3 cells growing at high density.
In Chapter 4, I describe the biochemical purification schemes I used to collect stimulatory fractions from high density supernates collected from HD ALL3 cells.
In Chapter 5, we focus on the biophysical study of the ALL3 cells in-vitro at low and high cell densities using atomic force microscopy (AFM). We measured several biomechanical parameters such as Young’s elastic modulus (measure of ‘cell’s stiffness), pericellular brush length, effective number of pericellular brush per unit area, and size of pericelluar brush at single cell level. We found several unexpected biophysical phenomenon that signifies the importance of defective cancer cell-cell communication on biomechanical properties of cells.
In Chapter 6, we report on biophysical differences between quiescent and proliferating CD34+ leukemic patient samples collected from four untreated chronic myeloid leukemic (CML) patients using the AFM technique. We observed substantial differences between quiescent and proliferating cells in terms of the Young’s elastic modulus, pericellular brush length and its grafting density at the single cell level.
In Chapter 7, I investigated the possible role of FAM129C (BCNP1) (Family member of protein 129 C/B cell Novel Protein 1) in signaling and cancer because it was the most highly up-regulated in the stimulated non-growing LD ALL3 cells. I performed several bioinformatics analyses to understand its role in cancer. I also investigated the function of BCNP1 in cellular signaling pathways. I found that PI3K inhibition and p38 MAPK activation leads to reduction in phosphorylation of BCNP1 at serine residues, suggesting that BCNP1 phosphorylation is PI3K and p38MAPK dependent and may be involved in cancer.
|Advisor:||Clarkson, Bayard D., Darie, Costel C.|
|Commitee:||Andreescu, Silvana, Katz, Evgeny, Lufkin, Thomas, Melman, Artem|
|School Location:||United States -- New York|
|Source:||DAI-B 77/09(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Biochemistry|
|Keywords:||Cancer, Collective behavior, Exosomes, Fam129c or bcnp1, Leukemia, Quorum sensing|
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