Tryptophane-205 of human topoisomerase I is essential for camptothecin inhibition of negative but not positive supercoil removal

Positive supercoils are introduced in cellular DNA in front of and negative supercoils behind tracking polymerases. Since DNA purified from cells is normally under-wound, most studies addressing the relaxation activity of topoisomerase I have utilized negatively supercoiled plasmids. The present report compares the relaxation activity of human topoisomerase I variants on plasmids containing equal numbers of superhelical twists with opposite handedness. We demonstrate that the wild-type enzyme and mutants lacking amino acids 1–206 or 191–206, or having tryptophane-205 replaced with a glycine relax positive supercoils faster than negative supercoils under both processive and distributive conditions. In contrast to wild-type topoisomerase I, which exhibited camptothecin sensitivity during relaxation of both negative and positive supercoils, the investigated N-terminally mutated variants were sensitive to camptothecin only during removal of positive supercoils. These data suggest different mechanisms of action during removal of supercoils of opposite handedness and are consistent with a recently published simulation study [Sari and Andricioaei (2005) Nucleic Acids Res., 33, 6621–6634] suggesting flexibility in distinct parts of the enzyme during clockwise or counterclockwise strand rotation

Assembly and structural analysis of a covalently closed nano-scale DNA cage

The inherent properties of DNA as a stable polymer with unique affinity for partner molecules determined by the specific Watson–Crick base pairing makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates for investigations of DNA-interacting enzymes. More recently, strategies for synthesis of more complex two-dimensional (2D) and 3D DNA structures have emerged. However, the building of such structures is still in progress and more experiences from different research groups and different fields of expertise are necessary before complex DNA structures can be routinely designed for the use in basal science and/or biotechnology. Here we present the design, construction and structural analysis of a covalently closed and stable 3D DNA structure with the connectivity of an octahedron, as defined by the double-stranded DNA helices that assembles from eight oligonucleotides with a yield of ∼30%. As demonstrated by Small Angle X-ray Scattering and cryo-Transmission Electron Microscopy analyses the eight-stranded DNA structure has a central cavity larger than the apertures in the surrounding DNA lattice and can be described as a nano-scale DNA cage, Hence, in theory it could hold proteins or other bio-molecules to enable their investigation in certain harmful environments or even allow their organization into higher order structures

microRNA global expression analysis and genomic profiling of the camptothecin-resistant T-ALL derived cell line CPT-K5: DOI: 10.14800/rd.441

The clinical use of the camptothecin (CPT) derivatives, topotecan and irinotecan, has had a significant impact on cancer therapy. However, acquired clinical resistance to these drugs is common, which greatly hampers their clinical efficacy. MicroRNAs (miRNAs) is an exciting novel class of endogenous non-coding RNAs that negatively regulate gene expression of up to 50% of the protein-coding genes at the post-translational level. Abnormal expression of miRNAs is associated with pathogenesis of cancer and is also implicated in anticancer drug resistance phenotypes. We used global expression analysis to examine for differential miRNA expression between the camptothecin-resistant cell line CPT-K5 and its parental CPT-sensitive RPMI-8402. In the CPT-K5 cell line 18 miRNAs were deregulated. Fifteen of these were down-regulated and three were up-regulated. The miRNA-193a-3p, miR-130a-3p, and miR-29c-3p were the most down-regulated miRNAs at 205.9-fold, 33.9-fold and 5.5-fold, respectively, while the miRNA let-7i-5p was the most up-regulated at 3.9-fold. We used subtraction BAC-based array CGH analysis to examine for genomic copy number changes. Only for the three most down-regulated miRNAs a positive correlation was found with genomic loss of their chromosomal regions in which they are encoded. Potential functional targets of the differentially expressed miRNAs were examined by searching the miRBase and miRTarBase databases. Recurrent KEGG pathways that theoretically could be affected by the deregulated miRNAs are lysine degradation, cell cycle, PI3K-Akt-, ERbB- and p53- signaling pathways. We show that the intracellular levels of several miRNAs are significantly deregulated upon acquisition of CPT resistance in the T-ALL derived cell line CPT-K5, and that genomic copy number changes is not a major cause of deregulation. In addition, the most deregulated miRNAs in our study have previously been described to be involved in various types of chemotherapeutic resistance, including the chemotherapeutics CPT, gefitinib and cisplatin in other cancer and cell types. Our study adds to the current knowledge of the mechanisms of acquired CPT resistance. Specific miRNAs may prove to be future targets to reverse or inhibit development of CPT resistance thereby providing means for a more effective treatment

Extreme hyperleukocytosis in a pediatric T-ALL patient with a rare translocation, t(7;19)(q35;p13), and submicroscopic deletions at 4q25, 7q33 and 10q23

Although childhood T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk disease the outcome can vary considerably. The varying outcomes suggest that unrecognized factors may contribute to disease progression. We report on a 2-year-old T-ALL patient presenting with a very short history of constipation and extreme hyperleukocytosis (WBC 882×109/L). In her leukemic cells we detected the very rare translocation t(7;19)(q35;p13) and LYL1 overexpression. Additionally, we detected submicroscopic deletions at 4q25, 7q33 and 10q23 by oligo-aCGH analysis. We suggest that LYL1 overexpression contributed to the leukemic state and propose that the observed microdeletions may have influenced to the rapid disease progression

Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study

Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR 4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1 IS and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance. </p