Dissecting the molecular mechanisms of cell division: A novel methylation equilibrium regulates spindle size in mitotic cells and Multidisciplinary high-throughput screening to discover novel anti-leukemia small molecule drugs
- Author(s): Xia, Xiaoyu
- Advisor(s): Torres, Jorge Z
- et al.
Mitotic spindle assembly is a highly complex and specifically orchestrated event that occurs once per cell division. It relies on a multitude of protein complexes, protein-protein interactions, and regulatory mechanisms. To date, many proteins that associate with microtubules and function in mitotic spindle assembly have been identified and characterized. A strategy in the treatment of cancer has been to inhibit cell division with antimitotics, a group of natural and synthetic small molecules that arrest cells in mitosis, induce apoptosis, and cause cell death. My research focuses on finding potential cancer therapeutics by identifying novel antimitotic proteins, and by developing anti-leukemia small molecule drugs.
Leucine carboxyl methyltransferase-1 (LCMT1) and protein phosphatase methylesterase-1 (PME-1) are essential enzymes that regulate the methylation of the protein phosphatase 2A catalytic subunit (PP2AC). LCMT1 and PME-1 have been linked to regulating cell growth and proliferation, but the underlying mechanisms have remained elusive. We show here an important role for an LCMT1-PME-1 methylation equilibrium in controlling mitotic spindle size. Depletion of LCMT1 or overexpression of PME-1 led to long spindles. In contrast, depletion of PME-1, pharmacological inhibition of PME-1 or overexpression of LCMT1 led to short spindles. Furthermore, perturbation of the LCMT1-PME-1 methylation equilibrium led to defective cell divisions, induction of apoptosis and reduced cell viability. Thus, we propose that the LCMT1-PME-1 methylation equilibrium is critical for regulating mitotic spindle size and thereby proper cell division.
Targeting of the leukemia proliferation cycle has been a successful approach to developing antileukemic therapies. However, drug-screening efforts to identify novel anti-leukemia agents have been hampered by the lack of a suitable high-throughput screening platform for suspension cells that does not rely on FACS analyses. We report the development of a novel leukemia cell-based high-throughput chemical screening platform for the discovery of cell cycle phase specific inhibitors that relies on the use of chemical cell cycle profiling. We have used this approach to analyze the cell cycle response of leukemia CCRF-CEM cells to each of 181,420 drug-like compounds. This approach yielded cell cycle phase specific inhibitors of leukemia cell proliferation. Further analyses of the top G2-phase and M-phase inhibitors identified a leukemia specific inhibitors (Leusin-1). Leusin-1 arrests cells in G2-phase and trigger an apoptotic cell death. More importantly, Leusin-1 was more active in acute lymphoblastic leukemia cells than other types of leukemia, non blood-born cancers, or normal cells. Leusin-1 represents novel leukemia specific inhibitors and could be used to develop new anti-leukemia therapies.