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.
Bai
Tao
T
Department of Obstetrics and Gynecology, Wakayama Medical University, Wakayama 641-0012, Japan.
Tanaka
Tetsuji
T
Yukawa
Kazunori
K
Umesaki
Naohiko
N
D000970:Antineoplastic Agents / Q000494:pharmacology*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D019175:DNA Methylation / Q000187:drug effects; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D064550:Death-Associated Protein Kinases; D000077209:Decitabine; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D002583:Uterine Cervical Neoplasms
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D006877:Hydroxamic Acids; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; C012589:trichostatin A; D000077209:Decitabine; D015254:DNA Modification Methylases; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D001374:Azacitidine; D002945:Cisplatin