RESUMO
OBJECTIVES: Mantle cell lymphoma (MCL) is one of the most heterogeneous lymphoid neoplasms with a variable course of disease. Although t(11;14)(q13;q32) is the hallmark of MCL resulting in cyclin D1 (CCND1) overexpression in 90% of patients, this is difficult to validate by immunohistochemistry. We hypothesised that SOX11 could be a robust molecular biomarker for MCL. METHODS: We have developed very sensitive and specific RT-qPCR assay employing a poly-A specific RT primer to circumvent contamination from gDNA caused by the intron-less nature of SOX11. RESULTS: We found a significant difference between the expression levels of SOX11 in patients with MCL at diagnosis (n = 21) and in healthy donors (n = 18) (blood: P < 0.0001; marrow: P = 0.0001). SOX11 expression of very low levels close to the assay sensitivity was detected in only 2 of 18 healthy donors, while low levels of CCND1 expression was observed in all blood and 12 of 13 marrow samples within the defined detection limit of Cq = 40. In spiking experiments of the GRANTA-519 MCL cell line into mononuclear cells from normal donor, the sensitivity of the SOX11 assay was found to be 2 × 10(-4) , while the sensitivity of the CCND1 assay was estimated to 2 × 10(-3) because of the normal background expression. In longitudinal sampling from patients with MCL the minimal residual disease (MRD) values based on the SOX11 expression mirrored the clinical disease development. CONCLUSION: This SOX11 RT-qPCR assay could be a useful tool for MRD monitoring in patients with MCL.
Assuntos
Biomarcadores Tumorais/genética , Linfoma de Célula do Manto/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Transcrição SOXC/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Estudos de Casos e Controles , Linhagem Celular Tumoral , Ciclina D1/genética , Primers do DNA/genética , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , RNA Mensageiro/genética , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e EspecificidadeRESUMO
The exon junction complex (EJC) is deposited onto spliced mRNAs and is involved in many aspects of mRNA function. We have recently reconstituted and solved the crystal structure of the EJC core made of MAGOH, Y14, the most conserved portion of MLN51, and the DEAD-box ATPase eIF4AIII bound to RNA in the presence of an ATP analog. The heterodimer MAGOH/Y14 inhibits ATP turnover by eIF4AIII, thereby trapping the EJC core onto RNA, but the exact mechanism behind this remains unclear. Here, we present the crystal structure of the EJC core bound to ADP-AIF(3), the first structure of a DEAD-box helicase in the transition-mimicking state during ATP hydrolysis. It reveals a dissociative transition state geometry and suggests that the locking of the EJC onto the RNA by MAGOH/Y14 is not caused by preventing ATP hydrolysis. We further show that ATP can be hydrolyzed inside the EJC, demonstrating that MAGOH/Y14 acts by locking the conformation of the EJC, so that the release of inorganic phosphate, ADP, and RNA is prevented. Unifying features of ATP hydrolysis are revealed by comparison of our structure with the EJC-ADPNP structure and other helicases. The reconstitution of a transition state mimicking complex is not limited to the EJC and eIF4AIII as we were also able to reconstitute the complex Dbp5-RNA-ADP-AlF(3), suggesting that the use of ADP-AlF(3) may be a valuable tool for examining DEAD-box ATPases in general.