A novel mechanism for acquired cisplatin-resistance: Suppressed translation of death-associated protein kinase mRNA is insensitive to 5-aza-2'-deoxycitidine and trichostatin in cisplatin-resistant cervical squamous cancer cells

The molecular mechanism for cisplatin (CDDP)-resistance of cancer cells has not yet been clarified, despite extensive studies. Here, we investigated whether death-associated protein (DAP) kinase, an apoptosis modulator, was involved in CDDP-resistance by examining the ME180 human cervical squamous cancer cell line and 6 monoclonal ME180-derived CDDP-resistant subclones. Co-treatment with CDDP and 5-aza-2'-deoxycytidine (5-aza-CdR), a demethylating agent, significantly enhanced the CDDP-sensitivities of the parent cells and CDDP-resistant subclones. Subsequent removal of 5-aza-CdR rapidly reversed the CDDP-sensitivity of the CDDP-resistant subclones to their original levels, whereas the parent cells retained the enhanced CDDP-sensitivity for at least 24 h. Quantitative RT-PCR revealed that the CDDP-resistant subclones expressed higher DNA methyltransferase (DNMT) mRNA levels than the parent cells, suggesting that increased DNMT expressions easily restored the CDDP-resistance of the CDDP-resistant subclones following 5-aza-CdR removal. Although the parent cells showed hypermethylation in the DAP kinase promoter region, corresponding methylated bands were not detected in 2 of the 6 CDDP-resistant subclones by methylation-specific PCR. All 6 CDDP-resistant subclones expressed higher DAP kinase mRNA levels than the parent cells, as evaluated by quantitative RT-PCR. Although DAP kinase protein expression was strongly suppressed in the parent cells and CDDP-resistant subclones, 5-aza-CdR treatment of the parent cells dose-dependently stimulated the DAP kinase protein expression, and this was synergistically enhanced by inhibiting histone deacetylation via trichostatin treatment in addition to 5-aza-CdR. However, DAP kinase protein expression in the CDDP-resistant subclones was not stimulated by treatment with 5-aza-CdR and/or trichostatin. These results indicate that post-transcriptional translation of DAP kinase mRNA is strongly suppressed and insensitive to treatment with 5-aza-CdR and trichostatin in the CDDP-resistant subclones established from ME180 human cervical squamous cancer cells. This CDDP-resistance is accompanied by molecular changes that disturb the post-transcriptional translation of the DAP kinase mRNA, and these molecular changes are transiently restored by demethylation.