Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biology

Supervisor

Timoshenko, Alexander V

Abstract

Galectins are a family of -galactoside-binding proteins involved in cell stress responses and differentiation. Galectins are multifunctional proteins widely studied in many cell models including acute myeloid leukemia HL-60 cells where they mediate numerous intra- and extracellular functions in response to many stress-inducing stimuli. O-GlcNAcylation is a dynamic post-translational modification implicated in the regulation of many cellular diseases including cancers. The O-GlcNAc mediated expression and secretion of galectins during neutrophilic differentiation was examined in HL-60 cells. Galectin gene (LGALS), galectin protein expression, and galectin secretion were determined by RT-qPCR, immunoblotting, and ELISA, respectively. Inhibition of O-GlcNAcylation induced markers of differentiation namely growth arrest, segmented nuclear morphology, and H2O2 production by NADPH oxidase comparable to ATRA-induced differentiation. The reduction of O-GlcNAcylation modified galectin expression, and increased galectin secretion in a manner similar to ATRA. This study confirms that inhibition of O-GlcNAcylation is one of the important mechanisms regulating expression and secretion of galectins and cellular differentiation.

Summary for Lay Audience

Galectins are a family of -galactoside-binding proteins widely studied in many cell models including HL-60 acute myeloid leukemia cells where they mediate many intra- and extracellular functions in response to many stress inducing stimuli. Acute myeloid leukemia (AML) is cancer occurring in immune cells of the myeloid lineage. Cancer is characterized as uncontrollable cell growth and division that results in the formation of tumors which negatively affect the normal cells and tissues nearby. Uncontrolled growth is often caused by gene mutations that can both upregulate genes that force cells through signaling pathways that promote growth and downregulate genes that keep cell division in check and suppress tumor formation. In AML, the cancer occurs in cells with more stem-like features that promote high growth and proliferation. These cells ordinarily will continue to develop and differentiate throughout their life cycle until they terminally differentiate into a cell type that can no longer divide. AML can be treated clinically using therapies that induce this terminal differentiation thus limiting tumor expansion. However, some differentiation therapies are not universally applicable therefore in this study, we sought to investigate the potential of O-GlcNAcylation modulation as a method of terminal differentiation. Many cancers display aberrant O-GlcNAcylation, a post-translational modification that regulates protein function, with many more proteins modified by this sugar in cancer cells than in healthy cells. In this study, I used ATRA to induce granulocytic differentiation of HL-60 cells and compares the results to those obtained by treatment biochemical inhibitors of enzymes that regulate O-GlcNAcylation to disrupt O-GlcNAc homeostasis. Reduction of protein O-GlcNAcylation led to slowed growth of AML cells, increased expression of galectins, and phagocytic activity similar to what is seen when cells are treated with established differentiation chemicals. Neutrophils induced by either ATRA, or inhibition of O-GlcNAc showed high expression levels of neutrophil-specific genetic markers, segmented nuclear morphology, and increased reactive oxygen species production compared to promyeloid cells, confirming that targeting O-GlcNAcylation can induce terminal differentiation. This provides novel insight into AML regulatory mechanisms and could be used to develop more utilitarian chemotherapies.

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