Silencing of astrin induces the p53-dependent apoptosis by suppression of HPV18 E6 expression and sensitizes cells to paclitaxel treatment in HeLa cells
Section snippets
Materials and methods
Antibodies. Purified 6× His-tagged recombinant astrin (a.a. 1–952) was prepared to immunize BALB/c mice. Spleens taken from immunized mice were gently ground and fused with myeloma cells. Positive hybridomas were determined by enzyme-linked immunosorbent assay (ELISA); successful clones were tested using immunoblotting and immunofluorescence analysis. Monoclonal antibodies against MDR, β-tubulin, and β-actin were purchased from Santa Cruz Biotech. (Santa Cruz, CA). Anti-p53 and anti-Bax
Generation of monoclonal antibody against human astrin
The specificity of the monoclonal anti-astrin antibody was determined by Western blot analysis using HeLa cell extracts. Two products with a molecular mass of 140 kDa were detected (Fig. 1A), indicating that astrin may have two splicing proteins. Similar results were observed in ovarian carcinoma ES2 cells and colon carcinoma HCT116 cells (data not shown). Immunofluorescent studies showed that astrin associated with microtubules in the cytoplasm of interphase cells and localized to the mitotic
Discussion
Our results show that astrin siRNA induces a p53-dependent apoptosis and highlights a potential role for human astrin in paclitaxel sensitivity. We also show that loss of astrin not only increases paclitaxel-induced cell death in parental HeLa cells but also in paclitaxel-resistant cells.
The tumor suppressor p53 plays a pivotal role in cancer development and can be activated by DNA damage, hypoxia, or aberrant oncogene expression to promote cell-cycle checkpoints, DNA repair, cellular
Acknowledgments
We are grateful to Drs. Mary Jeanne Buttrey, Chih-Long Chang, and Chin-Yuan Tzen for critical comments on this article. This work was partly supported by grants from National Science Council to YCY (NSC 94-2314-B-195-021) and MSC (NSC 94-2311-B-195-002).
References (38)
Paclitaxel-resistant human ovarian cancer cells have mutant β-tubulin that exhibit impaired paclitaxel-driven polymerization
J. Biol. Chem.
(1997)- et al.
Overexpression of ErbB2 blocks Paclitaxel-induced apoptosis by upregulation of p21Cip1, which inhibits p34Cdc2 kinase
Mol. Cell
(1998) - et al.
AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to paclitaxel
Cancer Cell
(2003) - et al.
Cloning and characterization of astrin, a new member of mitotic spindle-associated proteins
Biochem. Biophys. Res. Commun.
(2001) - et al.
Molecular interaction map of the p53 and Mdm2 logic elements, which control the off–on switch of p53 in response to DNA damage
Biochem. Biophys. Res. Commun.
(2005) - et al.
Molecular effects of paclitaxel: myths and reality
Int. J. Cancer
(1999) - et al.
Multiple microtubule alterations are associated with vinca alkaloid resistance in human leukemia cells
Cancer Res.
(2001) - et al.
Promotion of microtubule assembly in vitro by paclitaxel
Nature
(1979) - et al.
Paclitaxel stabilizes microtubules in mouse fibroblast cells
Proc. Natl. Acad. Sci. USA
(1980) - et al.
Paclitaxel and radiation
J. Natl. Cancer Int. Monog.
(1993)
Bcl2 is the guardian of microtubule integrity
Cancer Res.
Serine-70 is one of the critical sites for drug-induced Bcl2 phosphorylation in cancer cells
Cancer Res.
BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G2/M
Mol. Cell. Biol.
Mono- and multisite phosphorylation enhances Bcl2’s antiapoptotic function and inhibition of cell cycle entry functions
Proc. Natl. Acad. Sci. USA
p53 status does not affect sensitivity of human ovarian cancer cell lines to paclitaxel
Cancer Res
Disruption of p53 function in immortalized human cells does not affect survival or apoptosis after taxol or vincristine treatment
Clin. Cancer Res.
Mechanisms of paclitaxel resistance related to microtubules
Oncogene
Mechanisms of cancer drug resistance
Annu. Rev. Med.
Microtubule alterations and resistance to tubulin-binding agents
Int. J. Oncol.
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