Silencing of astrin induces the p53-dependent apoptosis by suppression of HPV18 E6 expression and sensitizes cells to paclitaxel treatment in HeLa cells

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Abstract

Astrin is a microtubule-associated protein and localizes with mitotic spindles in the M-phase. We silenced the expression of astrin protein and tested the cell viability in response to paclitaxel treatment in paclitaxel-sensitive and paclitaxel-resistant cells. We found that the absence of astrin by siRNA resulted in the activation of a p53-dependent apoptosis, which elevated pro-apoptotic Bax expression and increased the activity of caspase-3 in astrin-depleted cells. The HPV18 E6 transcription was found to be inhibited along with the increase expression of p53. Intriguingly, the expression of astrin decreased in paclitaxel-sensitive HeLa cells but remained steady in paclitaxel-resistant cells in response to paclitaxel treatment. Furthermore, we identified that the depletion of astrin caused more cell death both in paclitaxel-sensitive and -resistant cells in combination with paclitaxel treatment. These findings suggest that the silencing of astrin induce a p53-dependent apoptosis and has an additive effect on paclitaxel treatment.

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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).

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