Brown, Timothy Richard Paul; (1996) The regulation of gene expression during differentiation of the human monoblastic cell line U937. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Differentiation of the myeloid cell lineage towards a monocyte/macrophage has been studied using the monoblastoid cell line U937 as a model system. Treatment of these cells with various chemical agents, including retinoic acid (RA), 1α,25-dihydroxycholecalciferol (D3), phorbol ester (PMA) and cytokines, results in cessation of proliferation and the appearance of characteristics consistent with the monocyte/macrophage phenotype. Numerous changes in gene expression are observed, including upregulation of c-fgr mRNA and p55c-fgr, which is thought to have a role in the mature monocyte/macrophage. In the first part of this work I have analysed the mechanisms by which c-fgr gene expression is regulated during U937 cell differentiation. In the second part I have explored the roles of members of the RXR class of transcription factors in U937 cell differentiation. Following induction of differentiation, c-fgr gene transcription is activated from a myeloid- specific promoter upstream of an unmapped exon, M4. By isolation of a human genomic cosmid clone, exon M4 was mapped 11.1 kb upstream of the c-fgr coding exons. The myeloid promoter was characterised using a luciferase reporter gene in transient transfection assays which established that basal levels of transcription required sequences in the region -343 to -128 with respect to the transcriptional start site. Furthermore, the region -1211 to -772 was found to be responsive to PMA, inducing a 4-fold increase in luciferase activity. No promoter sequences responsive to TNFα in combination with D3 were found, suggesting that these agents direct transcription of the c-fgr gene via a mechanism that differs from the PMA response. It was determined that both RXR and RXRβ mRNAs are expressed in undifferentiated U937 cells, and that their expression is upregulated 8-fold and 3-fold, respectively, following PMA-induced differentiation. To study the role of the RXRα and RXRβ in regulating gene expression during myeloid differentiation, U937 cells were stably transfected with mammalian expression vectors that directed synthesis of either sense or antisense human RXRα RNA or antisense RXRβ RNA. It was shown that RXRα-antisense transfected cells were resistant to 9-cis RA, all-trans RA and D3-induced inhibition of proliferation. Furthermore, RXRα-sense transfected cells displayed increased sensitivity to RA. However, RXRα-sense expressing cells did not show increased sensitivity to D3, possibly as a result of limiting levels of vitamin D receptor. Both these novel cell lines could be induced to differentiate to the same extent as control cells by treatment with either TNFα or PMA, suggesting that these agents signal differentiation via different pathways to RA and D3. In contrast to these findings, RXRβ-anti-sense cells displayed no such resistance to any of the above treatments, suggesting that RXRβ performs different functions, possibly in more mature cells. The levels of β2-integrin cell surface antigens, CDlla, CDllb, CDllc and CD18, were not affected by expression of antisense RXR? mRNA compared to control cells, whereas sense expression of sense RXRα mRNA resulted in increased levels of the antigens. It was concluded that RXR?, but not RXR?, plays an important role in signalling the cascade of events that result in cessation of proliferation but not necessarily other aspects of monoblastic differentiation. Both aspects of this thesis have helped to delineate mechanisms of gene regulation that function during monoblastic differentiation.

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