Regulation of Myeloid Differentiation by c-Myc and Its Antagonists

Ryan, Kevin M (1996) Regulation of Myeloid Differentiation by c-Myc and Its Antagonists. PhD thesis, University of Glasgow.

Abstract

The enforced expression of c-myc is able to block the differentiation of myeloid cells. More recently, it has been shown that the correct functioning of c-Myc is not only dependent on the abundance of its dimerization partner, Max, but also on the levels of two other proteins which complex with Max, Mad and Mxil. Analysis was made of the levels of their mRNAs, relative to those of c-myc mRNA, during the induced differentiation of myeloid leukaemic HL60 and U937 cells. This revealed that, the abundance of mxil and max mRNA were largely maintained at levels comparable to those observed in untreated cells, but the levels of max mRNA were found to be markedly reduced at the very late stages of differentiation in HL60 cells induced by TPA. In contrast, the levels of mad mRNA were rapidly increased following differentiation induction by TPA. However, it was found that differentiation to granulocytes or monocytes/macrophages could also be achieved without a concomitant increase in the abundance of mad mRNA. To further investigate the role of Mad during the differentiation of myeloid cells E-box DNA-binding was analysed. While Myc:Max complexes were lost rapidly following differentiation induction, no Mad-containing complexes were detected during differentiation to monocytes/macrophages, and those which were detected during granulocytic differentiation were only evident at the very late stages. The subsequent analysis of these Mad-containing complexes revealed that they were also unlikely to be able to antagonise c-Myc function as they did not contain Max. In light of these findings, it was decided not to study these factors further, but to focus on the role played by c-myc in the control of differentiation per se. Although the mechanism by which c-Myc affects this process remains unknown, it is considered that it might result indirectly as an outcome of the continued cell-cycle progression invoked by c-Myc in cells which must growth arrest in order to differentiate. However, it is equally possible that a differentiation blockage occurs through a mechanism independent of c-Myc's involvement in cell-cycle progression. An analysis was therefore made of a differentiation-defective variant of the U937 cell line which, following treatment with TPA, does not differentiate, but rapidly ceases to proliferate, arresting at the G0/G1 phase of the cell cycle. Analysis during growth arrest revealed that, although this line down-regulated the expression of the Myc target gene, ornithine decarboxylase, it continued to express high levels of c-Myc protein, which retained the ability to bind its target sequence, CACGTG. Consequently, it was hypothesised that the continued expression of c-Myc in these cells may be responsible for their inability to differentiate in response to treatment with TPA. In agreement with this, down-regulation of the levels of c-Myc by antisense oligonucleotides directed against c-myc mRNA resulted in these cells acquiring characteristics of a terminally differentiated cell. As Mad, Max and Mxil have all been shown to antagonize c-Myc function, an analysis was also made for mutations in the genes for these proteins. Both HL60 and HeLa cells were found to be hemizygous for max. Sequencing of the remaining allele in these cell lines revealed three nucleotide changes, when compared to the published sequence. However, these changes did not result in any amino acid change. The relevance of all these findings to the regulation of myeloid differentiation, and in particular to the involvement of c-Myc in these processes, is discussed.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: George Birnie
• Keywords: Molecular biology
• Date of Award: 1996
• Depositing User: Enlighten Team
• Unique ID: glathesis:1996-75596
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 19:21
• Last Modified: 19 Nov 2019 19:21
• URI: https://theses.gla.ac.uk/id/eprint/75596