Convergence Of Proliferative And Survival Signals On The pRB/E2F Pathway In Haematopoietic Cells

Parad-Rajmankina, Yelena (2002). Convergence Of Proliferative And Survival Signals On The pRB/E2F Pathway In Haematopoietic Cells. MPhil thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000fbe6

Abstract

Chronic myeloid leukemia (CML) is a malignant stem cell disease characterised by an expansion of myeloid progenitor cells. These cells express a BCR/ABL fusion protein with constitutively activated tyrosine kinase activity, which causes a deregulation of apoptosis and cell cycle progression. In the pro-B BaF3 cell line, BCR/ABL has been shown to abrogate the EL-3 dependence to proliferate, but the signalling pathways activated by BCR/ABL and IL-3 to promote proliferation and survival are not yet well defined. In this study, BaF3 cells and a BaF3 cell line stably over-expressing BCR/ABL, BaF3-p210, were used to identify the downstream targets of BCR/ABL and IL-3. Both BCR/ABL and IL-3 were shown to induce the expression of cyclin D2 and inhibit the expression of the cell cycle inhibitor, p27 kipl. This regulation was shown to be directly due to BCR/ABL, in haematopoietic cells, by two different approaches. First, using a BaF3 cell line (TonB210.1) where the BCR/ABL expression is inducible by doxycycline and second, by inhibiting the kinase activity of BCR/ABL with the Abl tyrosine kinase inhibitor STI571. In order to establish the functional significance of cyclin D2 and p27 Kipl expression in response to IL-3 and BCR/ABL expression, the effects of ectopic expression of cyclin D2 and p27 Kipl on cell proliferation and survival were studied. The results demonstrate that both cyclin D2 and p27 Kipl have a role in BaF3 cell proliferation and survival, as ectopic expression of cyclin D2 is sufficient to abolish the cell cycle arrest and apoptosis induced by IL-3 withdrawal or BCR/ABL inactivation, while over-expression of p27 Kipl can cause cell cycle arrest and apoptosis in BaF3 cells. Next, the signal pathways triggered by BCR/ABL and IL-3 to regulate cell proliferation and apoptosis via cyclin D2 and p27 Kipl were investigated. The PI 3-Kinase inhibitor LY294002 blocks the ability of BCR/ABL or IL-3 to induce cyclin D2 up-regulation and p27 Kipl down-regulation and inhibits BCR/ABL-induced entry in S phase. Ectopic expression of cyclin D2 was found to overcome the cell cycle arrest induced by inhibition of PI 3-Kinase by LY294002. The results indicate that BCR/ABL and EL-3 target cyclin D2 and p27 Kipl to mediate cell cycle arrest and apoptosis through a pathway involving the phosphatidylinositol-3 kinase (PI 3-K).

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