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Blotting, respectively (Table three, Figure 7A). In four situations downregulation of DCK
In 4 situations downregulation of DCK gene BC-48Formula expression was observed in R compared to D samples (difference in CT (DCK-GAPDH) amongst R and D samples was > 1 cycle), while in 4 situations no modify was observed (difference in CT < 1 cycle) (Table 3). Western blotting analysis of the sample R2 compared to D2 revealed marked downregulation of protein DCK thereby confirming the gene expression results (i.e. 4-fold decrease in total DCK mRNA after araC-based therapy). Interestingly, protein DCK in the sample R6 compared PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25017212 to D6 was also moderately downregulated in spite of its gene expression remained practically unchanged (Figure 7A, Table three). As well as the analysis of MCL samples obtained from the relapsed sufferers, paired PF-06700841References principal cells isolated from two MCL sufferers (samples D9/R9, and D10/R10) refractory to araC have been topic to evaluation of gene and protein expression, and determination of their ex vivo sensitivity to nucleoside analogs (Figure 7B,C). The samples were obtained prior to araC administration (D9, D10), and 14 days soon after araC administration (R9, R10). Downregulation of both geneTable 2 Identity of differentially expressed proteins with low mascot score confirmed by MS/MSSpot no. 1 7 Accession P27707 P31937 Protein name Deoxycytidine kinase 3-Hydroxyisobutyrate Dehydrogenase, Mitochondrial Peptide sequence LKDAEKPVLFFER, QLCEDWEVVPEPVAR DFSSVFQFLREEETF (C-term), SPILLGSLAHQIYR Score 41, 46 49,Klanova et al. Molecular Cancer 2014, 13:159 http://www.molecular-cancer.com/content/13/1/Page 6 ofFigure three Western blot evaluation confirms marked downregulation of protein DCK in all R clones. Relative expression of deoxycytine kinase (DCK) in all 5 R and CTRL clones. Quadruplicate cell lysates had been separated on 12 SDS-PAGE minigels. Proteins were then transferred onto PVDF membranes, blocked and probed with anti-DCK antibody. Anti-GAPDH antibody was employed as the loading handle.and protein DCK expression was confirmed in R9 in comparison with D9 cells (Figure 7B). Sensitivity of R9 cells to araC, fludarabine and gemcitabine was substantially suppressed compared to D9 cells (Figure 7C). Each gene expression and protein expression of R10 in comparison with D10 sample remained unchanged (Figure 7B). Interestingly, susceptibility of R10 cells when compared with D10 cells to undergo apoptosis just after their ex vivo exposure to araC was improved in spite of the fact that R10 cells had been isolated just after administration of four cycles of high-dose araC (Figure 7C).Discussion In this study we analyzed molecular mechanisms of araC resistance in 5 MCL cell lines and ten paired primary MCL samples obtained ahead of and following araC-based therapies. Furthermore, we tested optimal treatment techniques for cytarabine-resistant MCL. On molecular level we identified marked and principal downregulation of DCK, the rate-limiting enzyme of nucleotide salvage pathway, in all five cytarabine-resistant MCL clones, and in 50 principal MCL samples obtained from patients, who progressed on or relapsed immediately after araC-based treatments. In 50 key MCL samples, no change of DCK expression was observed at time of lymphoma relapse or progression.Importantly, no upregulation of DCK was observed in any of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25486018 the analyzed post-treatment samples. Although the analysis from the principal MCL samples indicate that the mechanisms accountable for araC resistance in vivo are more complex than those observed in vitro, it has to be emphasized that downregulation of gene and protein DCK.Blotting, respectively (Table three, Figure 7A).
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