Cultivation and Differentiation Change Nuclear Localization of Chromosome Centromeres in Human Mesenchymal Stem Cells

<div><p>Chromosome arrangement in the interphase nucleus is not accidental. Strong evidences support that nuclear localization is an important mechanism of epigenetic regulation of gene expression. The purpose of this research was to identify differences in the localization of centromeres of chromosomes 6, 12, 18 and X in human mesenchymal stem cells depending on differentiation and cultivating time. We analyzed centromere positions in more than 4000 nuclei in 19 mesenchymal stem cell cultures before and after prolonged cultivation and after differentiation into osteogenic and adipogenic directions. We found a centromere reposition of HSAX at late passages and after differentiation in osteogenic direction as well as of HSA12 and HSA18 after adipogenic differentiation. The observed changes of the nuclear structure are new nuclear characteristics of the studied cells which may reflect regulatory changes of gene expression during the studied processes.</p></div

RDCs of HSA6, HSA12, HSA18 and HSAX, and the random signal distribution in the volume of the nucleus.

<p>RDCs of HSA6, HSA12 and HSA18 (A) significantly differ from random distribution (χ2 criterion, p = 10–9, p = 10–12 and p = 10–73 for HSA6, HSA12 and HSA18, respectively). For RDCs of HSAX in female and male cultures (B) the difference between random and non-random distribution was not significant (p = 0,09 and p = 0,3 for HSAX in female and male cells, respectively), however combined data from female and male cells gave statistical power to fix the expected differences (p = 0.03).</p

Statistical characteristics of RDCs of the HSA6, HSA12, HSA18 and HSAX.

<p>Statistical characteristics of RDCs of the HSA6, HSA12, HSA18 and HSAX.</p

RDCs of HSAX in male cells at early and late passages.

<p>RDCs of HSAX in male cells change after prolonged cultivation (p = 0.03). Dashed line demonstrates random distribution for comparison.</p

RDCs of HSA12 in MSC at early passages, after adipogenic differentiation and in lymphocytes.

<p>RDCs of HSA12 differ between MSC cells at early passages, after adipogenic differentiation (p = 0,008) and lymphocytes (p<10^–9).</p

Schematic distribution of RDCs of HSA12, HSA18, HSA6 и HSAX in MSC nucleus.

<p>The sizes of the colored circles show the frequency of finding the centromere in the particular radial interval. Braun—HSA6, yellow—HSA12, light-blue—HSA18, violet—HSAX (female), green—HSAX (male).</p

RDCs of HSAX in female cells before and after osteogenic differentiation.

<p>RDCs of HSAX in female cells change osteogenic differentiation of MSC (p = 0,04). Dashed line demonstrates random distribution for comparison.</p

Nuclear characteristics.

<p>The nucleus is counterstained with DAPI. The centromere signals are seen as red and green dots. The following characteristics of the nuclear are shown: r is the distance from the nuclear mass center (MC) to the center of the signal, R is radius passing through the center of the signal to the nuclear edge; L is the distance between centromeres; α—is the angle between the signals of two centromeres. We determined relative distance of the centromere (RDC) as r/R for each signal. The distance (L) between all pairs of signals and angels (alfa) were also measured.</p

Exome, transcriptome and miRNA analysis don’t reveal any molecular markers of TKI efficacy in primary CML patients

Abstract Background Approximately 5–20% of chronic myeloid leukemia (CML) patients demonstrate primary resistance or intolerance to imatinib. None of the existing predictive scores gives a good prognosis of TKI efficacy. Gene polymorphisms, expression and microRNAs are known to be involved in the pathogenesis of TKI resistance in CML. The aim of our study is to find new molecular markers of TKI therapy efficacy in CML patients. Methods Newly diagnosed patients with Ph+ CML in chronic phase were included in this study. Optimal and non-optimal responses to TKI were estimated according to ELN 2013 recommendation. We performed genotyping of selected polymorphisms in 62 blood samples of CML patients, expression profiling of 33 RNA samples extracted from blood and miRNA profiling of 800 miRNA in 12 blood samples of CML patients. Results The frequencies of genotypes at the studied loci did not differ between groups of patients with an optimal and non-optimal response to TKI therapy. Analysis of the expression of 34,681 genes revealed 26 differently expressed genes (p < 0.05) in groups of patients with different TKI responses, but differences were very small and were not confirmed by qPCR. Finally, we did not find difference in miRNA expression between the groups. Conclusions Using modern high-throughput methods such as whole-exome sequencing, transcriptome and miRNA analysis, we could not find reliable molecular markers for early prediction of TKI efficiency in Ph+ CML patients

Copy number variation analysis in cytochromes and glutathione S-transferases may predict efficacy of tyrosine kinase inhibitors in chronic myeloid leukemia

<div><p>Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the presence of <i>BCR/ABL</i> fusion gene in leukemic cells, which promotes uncontrolled cell proliferation. Up to 20% of CML patients show primary resistance or non-optimal response to tyrosine kinase inhibitor (TKI) therapy. We investigated the association between copy number variation (CNV) in glutathione S-transferases (GST) and cytochromes (CYP) and the response rate to TKI. We enrolled 47 patients with CML: 31 with an optimal response and 16 with failure at 6 months in accordance with European LeukemiaNet 2013 recommendations. CNV detection was performed using SALSA MLPA P128-C1 Cytochrome P450 probe mix. Patients with optimal response and with failure of TKI therapy showed different frequencies of wild type and mutated CYPs and GST (p<0.0013). Validation in the group of 15 patients proved high prognostic value (p = 0.02): positive and negative predictive value 83% and 78%; sensitivity and specificity 71% and 88%. Wild type genotypes of CYP and GST associate with a worse response to TKI treatment in CML patients. This test can be recommended for further clinical trials.</p></div