Tiwari, Sanjay; (1998) Cyclin dependent kinase regulation in haemopoietic cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Most stem cells are quiescent (Go) but have an extensive capacity to proliferate during the early stages of differentiation. These cells gradually lose their proliferative capacity along with differentiation and arrest in Go/G1 phase when released into the peripheral blood as mature cells. The regulation of the cell cycle during each step in haemopoiesis plays a critical role in the determination of cell fate. In particular, the molecular mechanisms which operate in G1 are crucial in regulating the transition of cells into, out of and through the cell cycle. The transition through mid/late G1 and entry into S phase is regulated by the activity of cyclin dependent kinases (CDKs). The overall aim of this project was to examine the regulation of these kinases in haemopoietic progenitor cells as they traverse into, out of and through the cell cycle. The first results chapter examines key components of the cell cycle machinery following stimulation of CD34+ cells to enter the cell cycle. Specifically, the kinetics of phosphorylation of the Retinoblastoma protein (pRb), which is involved in the regulation of G1 progression, was examined. In order to determine which CDKs may be involved in phosphorylating this substrate, the expression of cyclins and CDKs was analysed and the kinase activity of these CDKs was quantified. CDK6 is active early in G1 and is a prime candidate for the initial substrate phosphorylation. The activity of CDK4 is also detected in early G1 and cdk2 activity is detected as cells enter S phase. The regulation which occurs in exponentially proliferating cells differs from that in first time cycling cells. However, traditional cell synchronization methods perturb metabolic function giving rise to the possibility of unphysiological levels of protein expression. This chapter examines the expression of CDKs in exponentially proliferating cells using a novel three colour flow cytometric detection system. Changes in expression of specific cdks with respect to total cell protein content was determined in each cell cycle phase. Cyclins are shown to be expressed in only certain phases of the cell cycle, confirming previous findings. In contrast CDKs are expressed in all phases but they differ in their abundance during the cell cycle implying differential regulation between members of the CDK family. In addition, the regulation of cdc2 in proliferating CD34+ cells differs from that in proliferating T cells and this difference in regulation may have functional consequences on the differentiation process. The final results chapter examines growth arrest, a hallmark of memory T cell regulation. The induction of p21 and p27 inhibitors was examined in haemopoietic cells induced to growth arrest by a diverse array of inhibitory signals. The distinct patterns of induction of inhibitors demonstrate that inhibitory signaling pathways act independently and converge at some point in Gl.

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