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Farnesyl transferase inhibitors (FTIs) inhibit the farnesyl transferase (FT) enzyme, and have demonstrated effective anti-tumor activity with low toxicity in clinical trials; however, the molecular basis for their activity is not well defined. Farnesylation is a posttranslational modification that results in the addition of a farnesyl group (15-carbon chain) to the cysteine residue of proteins that harbor a carboxyl-terminal CAAX box. For example, the oncoprotein K-ras requires farnesylation for its plasma membrane localization and oncogenic signaling activity. Consequently, FTIs were developed as targeted agents that would prevent K-ras farnesylation by inhibiting FT; however, it was later shown that the anti-tumor activity of FTIs is independent of K-ras activation, suggesting the mechanism of FTI activity extends beyond inhibition of K-ras farnesylation. One possibility is the centromere-associated protein E (CENP-E), which is a farneslyated protein found on microtubules that is critical for proper chromosome alignment and segregation of the sister chromatids during mitosis. Here we used immunofluorescence to determine if CENP-E is affected by FTI treatment by staining for CENP-E, microtubules, and DNA. Specifically, we observed that 1A9 ovarian carcinoma cells treated with the FTI, lonafarib (LNF), for 16 hours lacked CENP-E localization at the kinetochore of the chromosome. In conjunction, chromosomes were mislocalized during metaphase of mitosis whereby a subset of chromosomes were found at the microtubule spindle poles. This was validated in 19 cancer cell lines, where it was observed that, the A549 lung cancer and 1A9 cancer cell lines had the highest percentage of mitotic cells with this chromosome migration defect (24% and 23%, respectively). To further evaluate this observation, we used live-cell confocal imaging to observe chromosome migration in real time. A549 cells transiently expressing Histone2B (H2B)-GFP plasmid (to allow for visualization of chromosomes) were treated with 1µM LNF. A delayed mitotic progression in prometaphase was observed, along with unaligned chromosomes. Herein, we have demonstrated that cancer cells treated with LNF do indeed result in abnormal chromosome migration to the metaphase plate, suggesting that FTIs can alter CENP-E function in cells.
• The FTI, lonafarnib (SCH66336), inhibits the farnesyl transferase enzyme (IC50 =1.5nM; Njoroge, FG et al., 1998)
• Lonafarnib inhibits the farnesylation of centromere associated protein-E (CENP-E) (Kirschmeier, P et al., 2000).
• CENP-E is critical for efficient capture and attachment of spindle microtubules by the kinetochore, which is required for proper chromosome alignment and segregation of the sister chromatids during mitosis.
• Studies using siRNA to knockdown CENP-E resulted in delayed mitotic progression, due to the appearance of unaligned chromosomes (Schebye, XM et al 2004). Studies have also shown that cells treated with FTIs alter the association of CENP-E with microtubules in vitro, but these affects were not observed in live cells, and chromosome localization patterns throughout mitosis were never assessed (Kirschmeier, P 2000).
• LNF delays mitotic progression in prometaphase
• Certain human cancer cell lines treated with LNF seem to be more sensitive to the disruption of chromosome migration during mitosis.
• Overall, LNF does indeed result in abnormal chromosome migration to the metaphase plate, suggesting that FTIs can alter CENP-E function in cells.
Future Directions
• Develop a vector expressing mutant CENP-E protein that is unable to be farneslyated.
• Cell cycle analysis will be used to determine if LNF induces a G2M or G1 pause in various human cancer cell lines.
• Investigate the structural relationship between the CENP-E farnesyl group and microtubule interactions.
Grant: NRSA (1 F32 CA108312-01A1) awarded to Adam I. Marcus
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