Low-dose cytarabine (LDAC) is a standard therapy for elderly acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. While high doses of cytarabine induce cytotoxicity, the precise ...mechanism of action of LDAC in AML remains elusive.
studies have demonstrated LDAC-induced differentiation; however, such differentiation is seldom observed
. We hypothesize that this discrepancy may be attributed to the influence of bone marrow (BM) stromal cells on AML cells. Thus, this study aimed to investigate the impact of BM stromal cells on LDAC-induced differentiation of AML cell lines and primary samples. Our results demonstrate that the presence of MS-5 stromal cells prevented LDAC-induced cell cycle arrest, DNA damage signaling and differentiation of U937 and MOLM-13 cell lines. Although transcriptomic analysis revealed that the stroma reduces the expression of genes involved in cytokine signaling and oxidative stress, data obtained with pharmacological inhibitors and neutralizing antibodies did not support the role for CXCL12, TGF-β1 or reactive oxygen species. The presence of stromal cells reduces LDAC-induced differentiation in primary samples from AML-M4 and myelodysplastic syndrome/AML patients. In conclusion, our study demonstrates that BM stroma reduces differentiation of AML induced by LDAC. These findings provide insights into the limited occurrence of terminal differentiation observed in AML patients, and suggest a potential explanation for this observation.
Acute myeloid leukemia (AML) is characterized by arrested differentiation making differentiation therapy a promising treatment strategy. Recent success of inhibitors of mutated isocitrate ...dehydrogenase (IDH) invigorated interest in differentiation therapy of AML so that several new drugs have been proposed, including inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme in pyrimidine synthesis. Cytarabine, a backbone of standard AML therapy, is known to induce differentiation at low doses, but the mechanism is not completely elucidated. We have previously reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) and brequinar, a DHODH inhibitor, induced differentiation of myeloid leukemia by activating the ataxia telangiectasia and Rad3-related (ATR)/checkpoint kinase 1 (Chk1) via pyrimidine depletion. In this study, using immunoblotting, flow cytometry analyses, pharmacologic inhibitors and genetic inactivation of Chk1 in myeloid leukemia cell lines, we show that low dose cytarabine induces differentiation by activating Chk1. In addition, cytarabine induces differentiation ex vivo in a subset of primary AML samples that are sensitive to AICAr and DHODH inhibitor. The results of our study suggest that leukemic cell differentiation stimulated by low doses of cytarabine depends on the activation of Chk1 and thus shares the same pathway as pyrimidine synthesis inhibitors.
All-trans retinoic acid (ATRA)-based treatment of acute promyelocytic leukemia (APL) is the most successful pharmacological treatment of acute myeloid leukemia (AML). Recent development of inhibitors ...of mutated isocitrate dehydrogenase and dihydroorotate dehydrogenase (DHODH) has revived interest in differentiation therapy of non-APL AML. Our previous studies demonstrated that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) induced differentiation of monocytic cell lines by activating the ATR/Chk1 via pyrimidine depletion. In the present study, the effects of AICAr on the viability and differentiation of primary AML blasts isolated from bone marrow of patients with non-APL AML were tested and compared with the effects of DHODH inhibitor brequinar and ATRA.
Bone marrow samples were obtained from 35 patients and leukemia blasts were cultured ex vivo. The cell viability was assessed by MTT assay and AML cell differentiation was determined by flow cytometry and morphological analyses. RNA sequencing and partial data analysis were conducted using ClusterProfiler package. Statistical analysis was performed using GraphPad Prism 6.0.
AICAr is capable of triggering differentiation in samples of bone marrow blasts cultured ex vivo that were resistant to ATRA. AICAr-induced differentiation correlates with proliferation and sensitivity to DHODH inhibition. RNA-seq data obtained in primary AML blasts confirmed that AICAr treatment induced downregulation of pyrimidine metabolism pathways together with an upregulation of gene set involved in hematopoietic cell lineage.
AICAr induces differentiation in a subset of primary non-APL AML blasts, and these effects correlate with sensitivity to a well-known, potent DHODH inhibitor.
All-
trans
retinoic acid (ATRA)-based therapy for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), is the most successful example of differentiation therapy. Although ...ATRA can induce differentiation in some non-APL AML cell lines and primary blasts, clinical results of adding ATRA to standard therapy in non-APL AML patients have been inconsistent, probably due to use of different regimens and lack of diagnostic tools for identifying which patients may be sensitive to ATRA. In this study, we exposed primary blasts obtained from non-APL AML patients to ATRA to test for differentiation potential in vitro. We observed increased expression of differentiation markers, indicating a response to ATRA, in four out of fifteen primary AML samples. Three samples in which CD11b increased in response to ATRA had an inversion of chromosome 16 as well as the CBFB-MYH11 fusion gene, and the fourth sample was from a patient with
KMT2A
-rearranged, therapy-related AML. In conclusion, we identified a subgroup of non-APL AML patients with inv(16) and CBFB-MYH11 as the most sensitive to ATRA-mediated differentiation in vitro, and our results can help identify patients who may benefit from ATRA treatment.
5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr) has been one of the most commonly used pharmacological modulators of AMPK activity. The majority of early studies on the role of AMPK, both in ...the physiological regulation of metabolism and in cancer pathogenesis, were based solely on the use of AICAr as an AMPK-activator. Even with more complex models of AMPK downregulation and knockout being introduced, AICAr remained a regular starting point for many studies focusing on AMPK biology. However, there is an increasing number of studies showing that numerous AICAr effects, previously attributed to AMPK activation, are in fact AMPK-independent. This review aims to give an overview of the present knowledge on AMPK-dependent and AMPK-independent effects of AICAr on metabolism, hypoxia, exercise, nucleotide synthesis, and cancer, calling for caution in the interpretation of AICAr-based studies in the context of understanding AMPK signaling pathway.
Metabolic pathways play important roles in proliferation and differentiation of malignant cells. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr), a precursor in purine biosynthesis and a ...well-established activator of AMP-activated protein kinase (AMPK), induces widespread metabolic alterations and is commonly used for dissecting the role of metabolism in cancer. We have previously reported that AICAr promotes differentiation and inhibits proliferation of myeloid leukemia cells. Here, using metabolic assays, immunoblotting, flow cytometry analyses, and siRNA-mediated gene silencing in leukemia cell lines, we show that AICAr-mediated differentiation was independent of the known metabolic effects of AMPK, including glucose consumption, but instead depends on the activation of the DNA damage–associated enzyme checkpoint kinase 1 (Chk1) induced by pyrimidine depletion. LC/MS/MS metabolomics analysis revealed that AICAr increases orotate levels and decreases uridine monophosphate (UMP) levels, consistent with inhibition of UMP synthesis at a step downstream of dihydroorotate dehydrogenase (DHODH). AICAr and the DHODH inhibitor brequinar had similar effects on differentiation markers and S-phase arrest, and genetic or pharmacological Chk1 inactivation abrogated both of these effects. Our results delineate an AMPK-independent effect of AICAr on myeloid leukemia differentiation that involves perturbation of pyrimidine biosynthesis and activation of the DNA damage response network.
Four filicide cases that occurred in Croatia in 2014-17 received wide national media coverage and caused strong public reactions. In each case, the mother of the child(ren) was the perpetrator.
This ...paper will analyse different representations of motherhood, the contradictions and inconsistencies between motherhood as an idea and ideology and everyday maternal practices and behaviours, with special attention being paid to the concept of maternal ambivalence and the expression of violent impulses towards children. Furthermore, the research will also paint a broader picture of the ideology and representations of motherhood. This will include the prescriptive norms of the ideal motherhood present in the public discourse, encompassing the official expert discourse of parenting, as well as the ‘private’ discourse of internet forums covering the issues of parenting and child rearing.
The purpose of this analysis is to show how the public profile of the mothers who committed filicide is created on the dichotomies of the ideal, self-abnegating mother and the monstrous ‘non-mother’ (Croatian: nemajka), with all of its horrifying, dehumanising characteristics, and how this influences the general construct of maternity, motherhood and the figure of the ideal mother in the patriarchal family and social structures.
A novel impregnation process for the fabrication of cotton nanocomposite with strong antimicrobial activity against antibiotics‐resistant bacteria and yeast was developed. The impregnation process ...includes the sol–gel treatment of fabric with (3‐aminopropyl)triethoxysilane in the first step, and synthesis of the CuO/Cu2O nanoparticles (NPs) on the fabric surface in the second step. The in situ synthesis of the CuO/Cu2O NPs was based on the adsorption of Cu2+‐ions by the introduced amino groups of the sol–gel coating. The adsorbed Cu2+‐ions are subsequently reduced in the alkaline solution of NaBH4. X‐ray diffraction measurements confirmed the formation of CuO/Cu2O NPs. Scanning electron microscopy and atomic absorption spectrometry analyses indicate that the particle size, agglomeration, and amounts of synthesized NPs were highly affected by the initial concentration of CuSO4 solution. The toxicity of nanocomposites to human keratinocytes (HaCaT) and antimicrobial activity against Gram‐negative Escherichia coli ATCC 25922, E. coli ATCC BAA 2469, and Klebsiella pneumoniae ATCC BAA 2146, and Gram‐positive bacteria Staphylococcus aureus ATCC 25923, S. aureus ATCC 43300 and yeast Candida albicans ATCC 24433 strongly depended on the copper content. In addition to excellent antimicrobial activity, controlled release of Cu2+‐ions from the fabrics into physiological saline solution was obtained.
Human pluripotent stem cells (hPSCs) provide an invaluable tool for modeling diseases and hold promise for regenerative medicine. For understanding pluripotency and lineage differentiation ...mechanisms, a critical first step involves systematically cataloging essential genes (EGs) that are indispensable for hPSC fitness, defined as cell reproduction in this study. To map essential genetic determinants of hPSC fitness, we performed genome-scale loss-of-function screens in an inducible Cas9 H1 hPSC line cultured on feeder cells and laminin to identify EGs. Among these, we found FOXH1 and VENTX, genes that encode transcription factors previously implicated in stem cell biology, as well as an uncharacterized gene, C22orf43/DRICH1. hPSC EGs are substantially different from other human model cell lines, and EGs in hPSCs are highly context dependent with respect to different growth substrates. Our CRISPR screens establish parameters for genome-wide screens in hPSCs, which will facilitate the characterization of unappreciated genetic regulators of hPSC biology.
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•A robust and flexible platform for genetic screens in hPSCs•Essential gene profiles for hPSCs on two substrates and core hPSC fitness gene sets•Previously unidentified regulators of hPSC fitness (DRICH1 and others)•Confirmation of the TP53 pathway, PAWR, PMAIP1 as negative proliferation regulators
Mair et al. establish a robust, inducible CRISPR screening platform for forward genetics in human pluripotent stem cells (hPSCs). Genome-wide proliferation screens identified core essential genes for hPSCs and revealed context-dependent genetic requirements on different substrates. This underlines hPSC plasticity and helps us to understand the genetic wiring of hPSCs.
Sustainable biodegradation of cellulose fibers is critical for composting after the end of a product’s life. In this study, we aimed at investigating the effect of in situ synthesized CuO/Cu
2
O ...nanoparticles (NPs) with biocidal concentration on the biodegradation behavior of cotton fibers pretreated with 1,2,3,4-butanetetracarboxylic acid (BTCA) and succinic acid (SUC). Biodegradation of the fibers was evaluated by soil burial tests in garden soil and in model compost after different soil burial times. The results showed that the application of BTCA, SUC, and CuO/Cu
2
O NPs did not affect the hydrophilicity of the samples and allowed a smooth biodegradation process. The morphological and chemical changes during biodegradation, evaluated by FESEM and FTIR analyses, showed that the presence of CuO/Cu
2
O NPs slightly hindered biodegradation of the fibers after 18 days in soil. However, biodegradation was much faster in the model compost, where all samples, regardless of their chemical modification, almost completely degraded after only 11 days. Intense microbial growth on the surface of all samples after nine days of burial in garden soil and model compost was confirmed by the presence of proteins produced by the microorganisms. The total number of microorganisms in the garden soil remained almost unchanged and increased in the model compost after the burial test. The only exception was the sample with the highest concentration of CuO/Cu
2
O NPs, which caused a reduction in microbial growth but not complete growth inhibition. These results clearly showed that during material degradation, the cellulosic material supporting microbial growth prevailed over the suppression of microbial growth by CuO/Cu
2
O NPs.
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