A comparative proteomic study identified LRPPRC and MCM7 as putative actors in imatinib mesylate cross-resistance in Lucena cell line

<p>Abstract</p> <p>Background</p> <p>Although chronic myeloid leukemia (CML) treatment has improved since the introduction of imatinib mesylate (IM), cases of resistance have been reported. This resistance has been associated with the emergence of multidrug resistance (MDR) phenotype, as a BCR-ABL independent mechanism. The classic pathway studied in MDR promotion is ATP-binding cassette (ABC) family transporters expression, but other mechanisms that drive drug resistance are largely unknown. To better understand IM therapy relapse due to the rise of MDR, we compared the proteomic profiles of K562 and Lucena (K562/VCR) cells.</p> <p>Results</p> <p>The use of 2-DE coupled with a MS approach resulted in the identification of 36 differentially expressed proteins. Differential mRNA levels of <it>leucine-rich PPR motif-containing (LRPPRC) protein, minichromosome maintenance complex component 7 (MCM7) </it>and <it>ATP-binding cassette sub-family B (MDR/TAP) member 1 (ABCB1) </it>were capable of defining samples from CML patients as responsive or resistant to therapy.</p> <p>Conclusions</p> <p>Through the data presented in this work, we show the relevance of MDR to IM therapy. In addition, our proteomic approach identified candidate actors involved in resistance, which could lead to additional information on BCR-ABL-independent molecular mechanisms.</p

Human Induced Pluripotent Stem Cells from Basic Research to Potential Clinical Applications in Cancer

The human induced pluripotent stem cells (hiPSCs) are derived from a direct reprogramming of human somatic cells to a pluripotent stage through ectopic expression of specific transcription factors. These cells have two important properties, which are the self-renewal capacity and the ability to differentiate into any cell type of the human body. So, the discovery of hiPSCs opens new opportunities in biomedical sciences, since these cells may be useful for understanding the mechanisms of diseases in the production of new diseases models, in drug development/drug toxicity tests, gene therapies, and cell replacement therapies. However, the hiPSCs technology has limitations including the potential for the development of genetic and epigenetic abnormalities leading to tumorigenicity. Nowadays, basic research in the hiPSCs field has made progress in the application of new strategies with the aim to enable an efficient production of high-quality of hiPSCs for safety and efficacy, necessary to the future application for clinical practice. In this review, we show the recent advances in hiPSCs&apos; basic research and some potential clinical applications focusing on cancer. We also present the importance of the use of statistical methods to evaluate the possible validation for the hiPSCs for future therapeutic use toward personalized cell therapies

Human Induced Pluripotent Stem Cells from Basic Research to Potential Clinical Applications in Cancer

The human induced pluripotent stem cells (hiPSCs) are derived from a direct reprogramming of human somatic cells to a pluripotent stage through ectopic expression of specific transcription factors. These cells have two important properties, which are the self-renewal capacity and the ability to differentiate into any cell type of the human body. So, the discovery of hiPSCs opens new opportunities in biomedical sciences, since these cells may be useful for understanding the mechanisms of diseases in the production of new diseases models, in drug development/drug toxicity tests, gene therapies, and cell replacement therapies. However, the hiPSCs technology has limitations including the potential for the development of genetic and epigenetic abnormalities leading to tumorigenicity. Nowadays, basic research in the hiPSCs field has made progress in the application of new strategies with the aim to enable an efficient production of high-quality of hiPSCs for safety and efficacy, necessary to the future application for clinical practice. In this review, we show the recent advances in hiPSCs’ basic research and some potential clinical applications focusing on cancer. We also present the importance of the use of statistical methods to evaluate the possible validation for the hiPSCs for future therapeutic use toward personalized cell therapies

Computational modeling of the bHLH domain of the transcription factor TWIST1 and R118C, S144R and K145E mutants

Abstract Background Human TWIST1 is a highly conserved member of the regulatory basic helix-loop-helix (bHLH) transcription factors. TWIST1 forms homo- or heterodimers with E-box proteins, such as E2A (isoforms E12 and E47), MYOD and HAND2. Haploinsufficiency germ-line mutations of the twist1 gene in humans are the main cause of Saethre-Chotzen syndrome (SCS), which is characterized by limb abnormalities and premature fusion of cranial sutures. Because of the importance of TWIST1 in the regulation of embryonic development and its relationship with SCS, along with the lack of an experimentally solved 3D structure, we performed comparative modeling for the TWIST1 bHLH region arranged into wild-type homodimers and heterodimers with E47. In addition, three mutations that promote DNA binding failure (R118C, S144R and K145E) were studied on the TWIST1 monomer. We also explored the behavior of the mutant forms in aqueous solution using molecular dynamics (MD) simulations, focusing on the structural changes of the wild-type versus mutant dimers. Results The solvent-accessible surface area of the homodimers was smaller on wild-type dimers, which indicates that the cleft between the monomers remained more open on the mutant homodimers. RMSD and RMSF analyses indicated that mutated dimers presented values that were higher than those for the wild-type dimers. For a more careful investigation, the monomer was subdivided into four regions: basic, helix I, loop and helix II. The basic domain presented a higher flexibility in all of the parameters that were analyzed, and the mutant dimer basic domains presented values that were higher than the wild-type dimers. The essential dynamic analysis also indicated a higher collective motion for the basic domain. Conclusions Our results suggest the mutations studied turned the dimers into more unstable structures with a wider cleft, which may be a reason for the loss of DNA binding capacity observed for in vitro circumstances.</p

Molecular functions altered by FoxM1 inhibition.

<p> <i>Data from IPA software (Ingenuity Systems).</i></p

Forkhead Box M1 (FoxM1) Gene Is a New STAT3 Transcriptional Factor Target and Is Essential for Proliferation, Survival and DNA Repair of K562 Cell Line

<div><p>The forkhead box (Fox) M1 gene belongs to a superfamily of evolutionarily conserved transcriptional regulators that are involved in a wide range of biological processes, and its deregulation has been implicated in cancer survival, proliferation and chemotherapy resistance. However, the role of FoxM1, the signaling involved in its activation and its role in leukemia are poorly known. Here, we demonstrate by gene promoter analysis, Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays that FoxM1 is a new target of the STAT3 transcriptional activator. Additionally, FoxM1 is transcriptionally dependent on STAT3 signaling activation. Furthermore, we verified that FoxM1 is crucial for K562 cell proliferation, cell cycle checkpoints and viability and could be related to chemotherapeutic resistance. By microarray analysis, we determined the signaling pathways related to FoxM1 expression and its role in DNA repair using K562 cells. Our results revealed new signaling involved in FoxM1 expression and its role in leukemic cells that elucidate cellular mechanisms associated with the development of leukemia and disease progression.</p> </div

Cell cycle and checkpoint genes are deregulated by FoxM1 inhibition.

<p>(A, B, C, D) Means (± standard deviation) of relative CCNB1, AURKA, SKP2, CDC25B mRNA levels of inhibition of K562 FoxM1 depleted cells at 24, 48 and 72 h. (E) Cell cycle content analysis (E), G1 phase, S phase, G2 phase, by Flow Cytometer. Two-way Anova test p<0,05.</p

STAT sites and STAT3 interaction with FoxM1 promoter.

<p>(A) 1000 bp sequence from FoxM1 promoter gene from start of transcription (+1), indicating STAT bindings sites (doted boxes), STAT3 binding sequence (*, listed boxes). Primer annealing region used to EMSA and ChIP analysis (arrows). (B) EMSA assay, free probe (FP), Nuclear protein extract (K562), Competidor oligo sequence (Comp), STAT3 oligo biding. STAT3 DNA complex (<−>), treatment using LLL-3 (LLL-3), nuclear protein extracts from untreated K562 cells (Unt). (C) ChIP assay, total DNA (Input), IgG and STAT3 immunoprecipiteated DNA (IgG and STAT3), non-template control (NTC).</p

Putative STAT binding sites on FoxM1 gene promoter.

*<p> <i>consensus sequence of STAT3 (TTMN_(4-6)DAA); M = A or C; D = A, G or T; N = any nucleotide. Transcriptional start site was determined by FoxM1 sequence (NM_202002).</i></p