RESUMO
Caspase-8 activation initiates apoptotic signaling cascades, and certain mutations in procasepase-8 have been reported to be associated with the progression and prognosis of different types of tumors. In this study, we have identified four novel mutations, which are highly correlated with chemotherapy resistance and poor prognosis of acute myeloid leukemia (AML) patients, within the P10 subunit of procaspase-8. These newly discovered mutations cause premature termination of translation, resulting in truncated procaspase-8 protein, which is incapable of forming dimer to initiate apoptosis signaling pathway. Further biochemical analysis reveals that the segment of P10 subunit of procaspase-8 consisting of three amino acid residues from L491 to F493 is crucial for the formation of procaspase-8 interdimer, and the aberration of this segment disrupts the dimerization and consequently precludes the activation of caspase-8 and downstream apoptotic signaling pathway. Therefore, the patients with AML who bear these types of P10 mutations were more likely to develop chemotherapy resistance due to impaired apoptotic signaling in cellular system, leading to significantly reduced overall survival (OS) as compared with patients carrying no such types of P10 mutations. Taken together, these newly identified P10 mutations in procaspase-8 could be used as novel biomarkers for predicting response and survival of chemotherapy-treated AML patients, as well as potential therapeutic targets for medical intervention in the future.
Assuntos
Biomarcadores Tumorais/genética , Caspase 8/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Mutação , Idoso , Antineoplásicos Fitogênicos/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Sequência de Bases , Biomarcadores Tumorais/metabolismo , Caspase 8/metabolismo , Progressão da Doença , Etoposídeo/farmacologia , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Multimerização Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
OBJECTIVE: To establish a routine procedure for the detection of p53 mutations in hepatocellular carcinoma (HCC) surgical resections using the FASAY (functional analysis of separated alleles of p53 on yeast) procedure. METHODS: p53 status was analyzed by FASAY and cDNA sequencing in 50 cases of HCC. After the extraction of RNA from the frozen tumor and corresponding normal tissues, reverse transcription RT-PCR was carried out using these samples. The assay can detect mutations of p53 mRNA between codons 67 and 347 by the DNA-binding activity of the protein and reveal them as red colonies. RESULTS: Of the 50 specimens, 29 (58%) were positive (mutant) by FASAY. Sequencing analysis confirmed that all 29 FASAY positive tumors harbored mutations, and that no mutations were detectable in any FASAY negative tumors. In 29 p53 mutations, 22 mutations were point missense mutation, 5 were deletions and 2 were splicing mutations. A novel splice mutation on splice donor of intron 6 was reported, which could produce two different mRNAs, respectively using the nearest upstream and downstream recessive splice donor sites. CONCLUSION: FASAY is a sensitive method for detecting the various types of p53 mutations in HCC, suggesting that the yeast functional assay for the detection of p53 mutations may be essential for elucidating their clinical significance.
