Cell cycle analysis and synchronization of theXenopus laevis XL2 cell line: Study of the kinesin related protein XlEg5

Uzbekov, Rustem; Prigent, Claude; Arlot‐Bonnemains, Yannick

doi:10.1002/(sici)1097-0029(19990401)45:1<31::aid-jemt3>3.0.co;2-k

Cited by 18 publications

(10 citation statements)

References 55 publications

(62 reference statements)

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“…This is consistent with the fact that Eg5 is thought to only function in spindle assembly, when cells have entered mitosis (27,28). In fact, preliminary data indicate that in breast tumors there is a positive correlation between mitotic index and Eg5 gene expression levels (29).…”

Section: Discussionmentioning

confidence: 99%

Mitotic Kinesin Inhibitors Induce Mitotic Arrest and Cell Death in Taxol-resistant and -sensitive Cancer Cells

Marcus

Peters

Thomas

et al. 2005

Journal of Biological Chemistry

159

123

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Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.

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Section: Discussionmentioning

confidence: 99%

Mitotic Kinesin Inhibitors Induce Mitotic Arrest and Cell Death in Taxol-resistant and -sensitive Cancer Cells

Marcus

Peters

Thomas

et al. 2005

Journal of Biological Chemistry

159

123

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“…The next issue was to demonstrate its expression at the protein level. For Western blotting analysis, we prepared HeLa cell extracts from sparsely cultured asynchronous cells or cells enriched at late G 2 /M phase by treatment with nocodazole, as it has been reported that the protein level of several kinesins is elevated during mitosis (Uzbekov et al, 1999;Gordon and Roof, 2001;Mayr et al, 2007;Fontijn et al, 2001;Feine et al, 2007;Ganguly et al, 2008). The antiserum raised against the bacterial His-tag fusion protein of the KIF18B-specific Cterminal region (aa residues 462-842) detected no band in both extracts.…”

Section: Generation Of Kif18b-specfic Abmentioning

confidence: 99%

Cell cycle-regulated expression and subcellular localization of a kinesin-8 member human KIF18B

Lee

Kim

Park

et al. 2010

Gene

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“…Cell synchronization XL2 cells were synchronized according to the protocol of Uzbekov et al (Uzbekov et al, 1999) with modifications. After serum starvation for 24 hours, cells were incubated 30 hours in complete medium with 2 µg/ml aphidicolin, then released from the block by several washes with fresh complete medium.…”

Section: Xenopus Cultured Cellsmentioning

confidence: 99%

“…The fraction of mitotic cells was estimated by direct counting in a phase contrast microscope. The percentage of cells in G2 and G1 was calculated as described elsewhere (Uzbekov et al, 1999). Data from more than 22,000 cells were used for estimation of cell fraction composition.…”

Section: Xenopus Cultured Cellsmentioning

confidence: 99%

Nucleolar association of pEg7 and XCAP-E, two members ofXenopus laeviscondensin complex in interphase cells

Uzbekov

Timirbulatova

Watrin

et al. 2003

Journal of Cell Science

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Cell cycle dynamics and localization of condensins — multiprotein complexes involved in late stages of mitotic chromosome condensation —were studied in Xenopus laevis XL2 cell line. Western blot analysis of synchronized cells showed that the ratio of levels of both pEg7 and XCAP-E to β-tubulin levels remains almost constant from G1 to M phase. pEg7 and XCAP-E were localized to the mitotic chromosomes and were detected in interphase nuclei. Immunostaining for condensins and nucleolar proteins UBF,fibrillarin and B23 revealed that both XCAP-E and pEg7 are localized in the granular component of the nucleolus. Nucleolar labeling of both proteins is preserved in segregated nucleoli after 6 hours of incubation with actinomycin D (5 mg/ml), but the size of the labeled zone was significantly smaller. The data suggest a novel interphase function of condensin subunits in spatial organization of the nucleolus and/or ribosome biogenesis.

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Cell cycle analysis and synchronization of theXenopus laevis XL2 cell line: Study of the kinesin related protein XlEg5

Cited by 18 publications

References 55 publications

Mitotic Kinesin Inhibitors Induce Mitotic Arrest and Cell Death in Taxol-resistant and -sensitive Cancer Cells

Mitotic Kinesin Inhibitors Induce Mitotic Arrest and Cell Death in Taxol-resistant and -sensitive Cancer Cells

Cell cycle-regulated expression and subcellular localization of a kinesin-8 member human KIF18B

Nucleolar association of pEg7 and XCAP-E, two members ofXenopus laeviscondensin complex in interphase cells

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