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Mechanisms of fostriecin, AGM-1470 and taxol disruption of cell cycle progression and cell activation Tudan, Christopher Richard


Signal transduction pathways regulating cell growth and differentiation consist largely of protein kinases that act within restricted phases of the cell cycle prior to a checkpoint and are also necessary for monitoring the integrity of DNA, DNA replication, and cell proliferation versus differentiation. Aberrations in these processes can often lead to uncontrolled growth and de-differentiation, and ultimately a pathological state. To gain insight into the particular signalling proteins that are responsible for these aberrations that lead to a diseased state, this study focused on three potential therapeutic agents, fostriecin, AGM-1470 and Taxol, that have demonstrated anti-neoplastic, anti-arthritic and antiangiogenic activity, and that mediate their effects at different phases of the cell cycle. Because of fostriecin's growth inhibitory effect on proliferating cells, its mechanism of action was investigated with BHK-21 cells, as well as the immortalized cell lines Jurkat, HL-60, CEM and U937. It was observed to accelerate entry into mitosis based on premature chromosome condensation (PCC) and nuclear lamina breakdown. These events also transpired in the presence of the DNA replication inhibitor aphidicolin and the DNA damaging agent VM-26. Mitotic induction and abrogation of the DNA replication and damage G2 phase checkpoint following fostriecin incubation occurred although fostriecin inhibited p34cdc2 kinase activity and endogenous histone H1 hyperphosphorylation. Mitotic induction was also observed in the presence of fostriecin at the restrictive temperature in FT210 cells that contain a temperature-sensitive lesion in the cdc2 gene. I conclude that fostriecin acts within the S and G2 phases of the cell cycle (no effect was observed in G1 synchronized cells) by accelerating cells into a mitotic catastrophe while down-regulating p34cdc2 kinase, perhaps by inhibiting protein phosphatase 2A. An agent affecting G1 phase progression, AGM-1470, was studied in HUVEC, a primary endothelial cell line that has been used to gain insight into angiogenesis mechanisms. AGM-1470 was shown to inhibit HUVEC proliferation while in G1 phase. This was probably due to its inhibition of protein kinase C (PKC), which must occur in G1 phase in HUVEC for entry into S phase. AGM- 1470 inhibition of DNA synthesis occurred if cells were incubated for only 15 minutes followed by re-incubation in drug-free medium. Cyclin-dependent kinase 4 (Cdk4) activity was also inhibited and may have contributed to the G1 block. To determine if AGM-1470 inhibited mitogen-activated protein (MAP) kinase family members, and because AGM- 1470 has demonstrated anti-arthritic efficacy in vivo, further studies were conducted in neutrophils as cell activation has been correlated with MAP kinase activity. Taxol was concurrently studied in this system as it has been reported to affect G1 phase-associated signalling events, including MAP kinase activation, in non-differentiated cells. Furthermore, Taxol is currently undergoing pre-clinical studies as an anti-arthritic and pseudogout agent. Taxol failed to inhibit PKC in response to calcium pyrophosphate dihydrate (CPPD) crystals, f-Met-Leu-Phe (fMLP) or phorbol myristate acetate (PMA), yet it reduced neutrophil activation by each of these agents. Taxol inhibited MAP kinase activation only in response to CPPD crystals. In contrast to Taxol, AGM-1470 inhibited activation of PKC in neutrophils in response to CPPD crystals, fMLP or PMA, but inhibited neutrophil activation only in response to fMLP or PMA. AGM-1470 did not block MAP kinase activation in response to CPPD crystals, but as noted for neutrophil activation, inhibited MAP kinase activation by fMLP or PMA. In view of these findings, I conclude that there appears to be an alternate-signalling pathway for neutrophil activation that proceeds through MAP kinase. Wortmannin, a PI 3-kinase inhibitor known to inhibit neutrophil activation when pretreated with CPPD or fMLP, failed to inhibit PMA induced chemiluminescence and activation of p70 S6 kinase. This further provides evidence for the hypothesis that AGM-1470 regulates the MAP kinase pathway and that MAP kinase is utilized by stimulated neutrophils during oxidative response. [Scientific formulae used in this abstract could not be reproduced.]

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