Artikel
DNA damage-induced expression of p53 suppresses mitotic checkpoint kinase hMps1: the lack of this suppression in p53mut cells contributes to apoptosis
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Veröffentlicht: | 20. März 2006 |
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Introduction: DNA damage induced by the topoisomerase I inhibitor irinotecan (CPT-11) triggers in p53wt colorectal carcinoma cells a long-term cell cycle arrest and in p53mut cells a transient arrest followed by apoptosis (Magrini et al., Int. J. Cancer 2002, Bhonde et al., Oncogene 2005). The mechanism of the p53-independent apoptosis still remains largely unclear.
Materials and methods: Here we used five p53wt and five p53mut established colon carcinoma cell lines to identify gene expression alterations associated with apoptosis in p53mut cells after treatment with SN-38, the CPT-11 metabolite. Cell cycle distribution was analysed by FACS. Gene expression on mRNA level was analysed by oligonucleotide microarrays and RT-PCR. Protein expression was analysed by Western blot.
Results: All five p53wt cell lines underwent a tetraploid cell cycle arrest whereas all the p53mut cell lines underwent apoptosis after treatment with SN-38. Microarray analysis showed that, after treatment, 21 mitosis-related genes were expressed at least twofold stronger in the apoptosis-executing p53mut cells than in the cell cycle-arrested p53wt cells. One of the genes whose strong post-treatment expression was associated with apoptosis was the mitotic checkpoint kinase hMps1 (human ortholog of the yeast monopolar spindle 1 kinase). hMps1 mRNA and protein expression was suppressed by the treatment-induced as well as by the exogenous adenovirus-coded p53 protein. The direct suppression of hMps1 by siRNA or inhibition of its activity by a dominant-negative hMps1 partly suppressed apoptosis.
Conclusions: 1. High expression of mitotic genes in p53mut cells after SN-38-treatment contributes to DNA damage-induced apoptosis whereas their suppression in p53wt cells acts as a safeguard mechanism preventing mitosis initiation. 2. hMps1 kinase is one of the mitotic checkpoint proteins whose expression after DNA damage in p53mut cells activates the checkpoint and contributes to apoptosis.
Authors who also contributed to the work include: 1Dhatchana Moorthy and 2H.W. Mewes