RESUMEN
Although urine-based liquid biopsy has received considerable attention, there is a lack of a simple model to optimize assay parameters, including cell-free DNA (cfDNA) extraction, bisulfite modification, and bis-DNA recovery after conversion for methylation analysis in urine. The primary aim of this work was to establish a practical model by developing a quantitative methylation-sensitive PCR (qMS-PCR) assay for PAX2 based on hypermethylated PAX2 cfDNA that could be detected in healthy human urine. We first studied the methylation status of PAX2 in kidney tissues and whole blood, followed by an assessment of commercial kits for bisulfite conversion and bis-DNA recovery. Furthermore, we investigated the influence of urine storage and collection conditions on the preservation of methylated PAX2 in urine samples by qMS-PCR. As expected, PAX2 methylation was identified in urine but not in blood. Two commercial kits (CellCook and Zymo Research) had similar conversion efficiency and bis-DNA recovery. Urine storage for up to 5 days did not change PAX2 methylation estimates. Overall, cold storage of urine samples and the CellCook urine container maintained higher levels of methylated PAX2 compared to urine kept at room temperature and the conventional tubes, respectively. These findings highlight the importance of using the correct approaches/kits and optimizing experimental conditions as a diagnostic tool in the clinical setting. Our study provides insights on the development of urine-based liquid biopsy with DNA methylation as a universal biomarker.
Asunto(s)
Ácidos Nucleicos Libres de Células , Metilación de ADN , Ácidos Nucleicos Libres de Células/genética , ADN/análisis , Voluntarios Sanos , Humanos , Riñón/química , Biopsia Líquida , Factor de Transcripción PAX2/genéticaRESUMEN
Residence of cancer-propagating cells (CPCs) within preferential microenvironmental niches has a major part in evading therapy. However, the nature of niches involved and the mechanisms protecting CPCs remain largely unknown. We addressed these issues in mouse transplantation models of acute lymphoblastic leukemia (ALL). When the engrafted leukemic cells substantially damaged adjacent microenvironment in the bone marrow (BM), after chemotherapy small foci of CPCs were retained, surrounded by sheaths of supporting cells that comprise a protective niche. We investigated patients' BM biopsies and found evidence of a similar process in patients receiving induction therapy. The efficacy of chemotherapy was enhanced by interfering with the niche formation or function. We therefore identified a therapy-induced niche that protects CPCs.
Asunto(s)
Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Nicho de Células Madre/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Biopsia , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Citarabina/administración & dosificación , Citarabina/farmacología , Daunorrubicina/administración & dosificación , Daunorrubicina/farmacología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
<p><b>OBJECTIVE</b>To explore the relationship between the polymorphisms of DNA repair gene (XRCC1 194, 280 and 399) and the chromosomal damage induced by benzene.</p><p><b>METHODS</b>The chromosomal damage of the peripheral lymphocytes in 459 workers occupationally exposed to benzene and 88 non-exposed controls were detected with cytokinesis-block micronucleus (CBMN) assay. PCR-RFLP technique was used to measure polymorphisms in XRCC1 194, 280 and 399.</p><p><b>RESULTS</b>It was found that the MN frequency (2.12‰ ± 1.88‰) of the exposed group was significantly higher than that (1.19‰ ± 1.68‰) of the control group (P < 0.05), in the exposed group, the MN frequency (3.00‰ ± 2.76‰) of older workers (> 35 years) was significantly higher than that (2.02‰ ± 1.71‰) of younger workers (≤ 35 years) (P < 0.05). The effect of genetic polymorphisms of XRCC1 on CBMN was not found. The haplotypes AAA/BAA, AAB/AAB, ABA/ABA, ABB/ABB could associated with the increased frequencies of total micronucleus (P < 0.05).</p><p><b>CONCLUSION</b>Benzene exposure could result in chromosome damage. Age of workers and diplotypes of XRCC1 could associated with chromosomal damage induced by benzene.</p>