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Adaptive DNA amplification of synthetic gene circuit opens a way to overcome cancer chemoresistance.Proceedings of the National Academy of... Dec 2023Drug resistance continues to impede the success of cancer treatments, creating a need for experimental model systems that are broad, yet simple, to allow the...
Drug resistance continues to impede the success of cancer treatments, creating a need for experimental model systems that are broad, yet simple, to allow the identification of mechanisms and novel countermeasures applicable to many cancer types. To address these needs, we investigated a set of engineered mammalian cell lines with synthetic gene circuits integrated into their genome that evolved resistance to Puromycin. We identified DNA amplification as the mechanism underlying drug resistance in 4 out of 6 replicate populations. Triplex-forming oligonucleotide (TFO) treatment combined with Puromycin could efficiently suppress the growth of cell populations with DNA amplification. Similar observations in human cancer cell lines suggest that TFOs could be broadly applicable to mitigate drug resistance, one of the major difficulties in treating cancer.
Topics: Animals; Humans; DNA; Drug Resistance, Neoplasm; Genes, Synthetic; Oligonucleotides; Puromycin; Mammals; Neoplasms
PubMed: 38019857
DOI: 10.1073/pnas.2303114120 -
Discover Oncology Nov 2023To investigate the influence of β-arrestin2 on the docetaxel resistance in castration-resistant prostate cancer (CRPC) and elucidate the underlying molecular mechanisms.
PURPOSE
To investigate the influence of β-arrestin2 on the docetaxel resistance in castration-resistant prostate cancer (CRPC) and elucidate the underlying molecular mechanisms.
METHODS
PC3 and DU145 cells with stable β-arrestin2 overexpression and C4-2 cells with stable β-arrestin2 knockdown, were constructed via using lentivirus and puromycin selection. MTT and colony formation assays were carried out to investigate the effect of β-arrestin2 expression on the docetaxel resistance of CRPC cells. Glycolysis analysis was used to assess the glycolytic capacity modulated by β-arrestin2. GO enrichment analysis, gene set enrichment analysis and Spearman correlation test were carried out to explore the potential biological function and mechanism via using public data from GEO and TCGA. The expressions of PKM2, Phospho-PKM2, Phospho-ERK1/2 and hnRNP A1 were detected by western blot. Functional blocking experiments were carried out to confirm the roles of PKM2 and hnRNP A1 in the regulation of β-arrestin2's biological functions via silencing PKM2 or hnRNP A1 expression in cells with stable β-arrestin2 overexpression. Finally, nude mice xenograft models were established to confirm the experimental results of cell experiments.
RESULTS
β-Arrestin2 significantly decreased the sensitivity of CRPC cells to docetaxel stimulation, through enhancing the phosphorylation and expression of PKM2. Additionally, β-arrestin2 increased PKM2 phosphorylation via the ERK1/2 signaling pathway and induced PKM2 expression in a post-transcriptional manner through an hnRNP A1-dependent PKM alternative splicing mechanism, rather than by inhibiting its ubiquitination degradation.
CONCLUSION
Our findings indicate that the β-arrestin2/hnRNP A1/PKM2 pathway could be a promising target for treating docetaxel-resistant CRPC.
PubMed: 38019357
DOI: 10.1007/s12672-023-00740-0 -
Biomedicines Nov 2023Podocytes play a central role in glomerular diseases such as (idiopathic) nephrotic syndrome (iNS). Glucocorticoids are the gold standard therapy for iNS. Nevertheless,...
Podocytes play a central role in glomerular diseases such as (idiopathic) nephrotic syndrome (iNS). Glucocorticoids are the gold standard therapy for iNS. Nevertheless, frequent relapses are common. In children with iNS, steroid-sparing agents are used to avoid prolonged steroid use and reduce steroid toxicity. Levamisole is one of these steroid-sparing drugs and although clinical effectiveness has been demonstrated, the molecular mechanisms of how levamisole exerts its beneficial effects remains poorly studied. Apart from immunomodulatory capacities, nonimmunological effects of levamisole on podocytes have also been suggested. We aimed to elaborate on the effects of levamisole on human podocytes in iNS. RNA sequencing data from a human podocyte cell line treated with levamisole showed that levamisole modulates the expression of various genes involved in actin cytoskeleton stabilization and remodeling. Functional experiments showed that podocytes exposed to puromycin aminonucleoside (PAN), lipopolysaccharides (LPS), and NS patient plasma resulted in significant actin cytoskeleton derangement, reduced cell motility, and impaired cellular adhesion when compared to controls, effects that could be restored by levamisole. Mechanistic studies revealed that levamisole exerts its beneficial effects on podocytes by signaling through the glucocorticoid receptor and by regulating the activity of Rho GTPases. In summary, our data show that levamisole exerts beneficial effects on podocytes by stabilizing the actin cytoskeleton in a glucocorticoid receptor-dependent manner.
PubMed: 38002039
DOI: 10.3390/biomedicines11113039 -
Clinical and Translational Medicine Nov 2023Translation dysregulation plays a crucial role in tumourigenesis and cancer progression. Oncogenic translation relies on the stability and availability of tRNAs for...
BACKGROUND
Translation dysregulation plays a crucial role in tumourigenesis and cancer progression. Oncogenic translation relies on the stability and availability of tRNAs for protein synthesis, making them potential targets for cancer therapy.
METHODS
This study performed immunohistochemistry analysis to assess NSUN2 levels in thyroid cancer. Furthermore, to elucidate the impact of NSUN2 on anaplastic thyroid cancer (ATC) malignancy, phenotypic assays were conducted. Drug inhibition and time-dependent plots were employed to analyse drug resistance. Liquid chromatography-mass spectrometry and bisulphite sequencing were used to investigate the m C methylation of tRNA at both global and single-base levels. Puromycin intake and high-frequency codon reporter assays verified the protein translation level. By combining mRNA and ribosome profiling, a series of downstream proteins and codon usage bias were identified. The acquired data were further validated by tRNA sequencing.
RESULTS
This study observed that the tRNA m C methyltransferase NSUN2 was up-regulated in ATC and is associated with dedifferentiation. Furthermore, NSUN2 knockdown repressed ATC formation, proliferation, invasion and migration both in vivo and in vitro. Moreover, NSUN2 repression enhanced the sensitivity of ATC to genotoxic drugs. Mechanically, NSUN2 catalyses tRNA structure-related m C modification, stabilising tRNA that maintains homeostasis and rapidly transports amino acids, particularly leucine. This stable tRNA has a substantially increased efficiency necessary to support a pro-cancer translation program including c-Myc, BCL2, RAB31, JUNB and TRAF2. Additionally, the NSUN2-mediated variations in m5C levels and different tRNA Leu iso-decoder families, partially contribute to a codon-dependent translation bias. Surprisingly, targeting NSUN2 disrupted the c-Myc to NSUN2 cycle in ATC.
CONCLUSIONS
This research revealed that a pro-tumour m5C methyltransferase, dynamic tRNA stability regulation and downstream oncogenes, c-Myc, elicits a codon-dependent oncogenic translation network that enhances ATC growth and formation. Furthermore, it provides new opportunities for targeting translation reprogramming in cancer cells.
Topics: Humans; Thyroid Carcinoma, Anaplastic; Methyltransferases; Codon; Thyroid Neoplasms; RNA, Transfer; rab GTP-Binding Proteins
PubMed: 37983928
DOI: 10.1002/ctm2.1466 -
Planta Medica Feb 2024Astragaloside IV (AS-IV) has been shown to provide renal protection in various kidney injury models. However, the metabolic profile variation of AS-IV in pathological...
Astragaloside IV (AS-IV) has been shown to provide renal protection in various kidney injury models. However, the metabolic profile variation of AS-IV in pathological models is not well established. This study aims to explore the metabolic pathway of AS-IV in the classical puromycin aminonucleoside (PAN)-induced kidney injury in a rat model. Twelve Wistar rats were randomly divided into the AS-IV (CA) and the PAN+AS-IV (PA) treatment groups. PAN was injected by a single tail intravenous (i. v.) injection at 5 mg/100 g body weight, and AS-IV was administered intragastrically (i. g.) at 40 mg/kg for 10 days. Fecal samples of these rats were collected, and metabolites of AS-IV were detected by ultra-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) to explore the AS-IV metabolic pathway. The metabolic differences between the AS-IV and PAN+AS-IV groups were compared. A total of 25 metabolites were detected, and deglycosylation, deoxygenation, and methyl oxidation were found to be the main metabolic pathways of AS-IV . The abundance of most of these metabolites in the PAN+AS-IV group was lower than that in the AS-IV treatment group, and differences for seven of them were statistically significant. Our study indicates that AS-IV metabolism is affected in the PAN-induced kidney injury rat model.
Topics: Rats; Animals; Tandem Mass Spectrometry; Chromatography, High Pressure Liquid; Rats, Wistar; Puromycin; Saponins; Triterpenes
PubMed: 37931776
DOI: 10.1055/a-2186-3182 -
BMC Nephrology Oct 2023Nephrotic syndrome (NS) is characterized by renal sodium and water retention. The mechanisms are not fully elucidated.
INTRODUCTION
Nephrotic syndrome (NS) is characterized by renal sodium and water retention. The mechanisms are not fully elucidated.
METHODS
The NS rat model was established by single intraperitoneal injection of 100 mg/kg puromycin aminonucleoside (PAN). The plasma electrolyte level and urinary sodium excretion were monitored dynamically. The changes of some sodium transporters, including epithelial Na channel (ENaC), Na/H exchanger 3 (NHE3), Na-K-2Cl cotransporter 2 (NKCC2) and Na-Cl cotransporter (NCC) in renal cortex at different time points and the level of peripheral circulation factors were detected.
RESULTS
The urinary sodium excretion of the model group increased significantly on the first day, then decreased compared with the control group, and there was no significant difference between the model group and the control group on the 12th day. The changes of peripheral circulation factors were not obvious. Some sodium transporters in renal cortex increased in varying degrees, while NKCC2 decreased significantly compared with the control group.
CONCLUSIONS
The occurrence of NS edema may not be related to the angiotensin system. The decrease of urinary sodium excretion is independent of the development of albuminuria. During the 18 days of observation, it can be divided into three stages: sodium retention, sodium compensation, and simple water retention. The mechanism is related to the increased expression of α-ENaC, γ-ENaC, NHE3 and NCC in a certain period of time, the compensatory decrease of NKCC2 expression and the continuous increase of aquaporin 2 (AQP2) expression.
Topics: Rats; Animals; Nephrotic Syndrome; Puromycin Aminonucleoside; Sodium; Sodium-Hydrogen Exchanger 3; Aquaporin 2; Epithelial Sodium Channels; Kidney; Membrane Transport Proteins; Solute Carrier Family 12, Member 3; Water
PubMed: 37880610
DOI: 10.1186/s12882-023-03367-z -
Journal of Biological Rhythms Feb 2024Circadian-paced biological processes are key to physiology and required for metabolic, immunologic, and cardiovascular homeostasis. Core circadian clock components are...
Circadian-paced biological processes are key to physiology and required for metabolic, immunologic, and cardiovascular homeostasis. Core circadian clock components are transcription factors whose half-life is precisely regulated, thereby controlling the intrinsic cellular circadian clock. Genetic disruption of molecular clock components generally leads to marked pathological events phenotypically affecting behavior and multiple aspects of physiology. Using a transcriptional signature similarity approach, we identified anti-cancer protein synthesis inhibitors as potent modulators of the cardiomyocyte molecular clock. Eukaryotic protein translation inhibitors, ranging from translation initiation (rocaglates, 4-EGI1, etc.) to ribosomal elongation inhibitors (homoharringtonine, puromycin, etc.), were found to potently ablate protein abundance of REV-ERBα, a repressive nuclear receptor and component of the molecular clock. These inhibitory effects were observed both in vitro and in vivo and could be extended to PER2, another component of the molecular clock. Taken together, our observations suggest that the activity spectrum of protein synthesis inhibitors, whose clinical use is contemplated not only in cancers but also in viral infections, must be extended to circadian rhythm disruption, with potential beneficial or iatrogenic effects upon acute or prolonged administration.
Topics: Circadian Clocks; Circadian Rhythm; Protein Synthesis Inhibitors; Nuclear Receptor Subfamily 1, Group D, Member 1; Heart
PubMed: 37872767
DOI: 10.1177/07487304231202561 -
STAR Protocols Dec 2023Translation is a fundamental process of cellular behavior. Here, we present a protocol for measuring translation in Drosophila epithelial tissues using...
Translation is a fundamental process of cellular behavior. Here, we present a protocol for measuring translation in Drosophila epithelial tissues using O-propargyl-puromycin (OPP), a puromycin derivative. We detail steps for larval dissection, OPP incorporation, fixation, OPP labeling, immunostaining, and imaging. We also provide details of quantification analysis. Significantly, OPP addition to methionine-containing media enables polypeptide labeling in living cells. Here, we study wing imaginal discs, an excellent model system for investigating growth, proliferation, pattern formation, differentiation, and cell death. For complete details on the use and execution of this protocol, please refer to Lee et al. (2018), Ji et al. (2019), and Kiparaki et al. (2022)..
Topics: Animals; Drosophila; Imaginal Discs; Larva; Puromycin
PubMed: 37862174
DOI: 10.1016/j.xpro.2023.102653 -
Genes & Genetic Systems Oct 2023Polyglutamine (polyQ) diseases are rare autosomal-dominant neurodegenerative diseases associated with the expansion of glutamine-encoding triplet repeats in certain...
Polyglutamine (polyQ) diseases are rare autosomal-dominant neurodegenerative diseases associated with the expansion of glutamine-encoding triplet repeats in certain genes. To investigate the functional influence of repeat expansion on disease mechanisms, we applied a biallelic genome-engineering platform that we recently established, called Universal Knock-in System or UKiS, to develop a human cell trio, a set of three isogenic cell lines that are homozygous for two different numbers of repeats (first and second lines) or heterozygous for the two repeat numbers (third line). As an example of a polyQ disease, we chose spinocerebellar ataxia type 2 (SCA2). In a pseudodiploid human cell line, both alleles of the glutamine-encoding triplet repeat in the SCA2-causing gene, ataxin 2 or ATXN2, were first knocked in with a donor sequence encoding both thymidine kinase and either puromycin or blasticidin resistance proteins under dual drug selection. The knocked-in donor alleles were then substituted with a payload having either 22 or 76 triplet repeats in ATXN2 by ganciclovir negative selection. The two-step substitution and subsequent SNP typing and genomic sequencing confirmed that the SCA2-modeling isogenic cell trio was obtained: three clones of 22-repeat homozygotes, two clones of 22/76-repeat heterozygotes and two clones of 76-repeat homozygotes. Finally, RT-PCR and immunoblotting using the obtained clones showed that, consistent with previous observations, glutamine tract expansion reduced transcriptional and translational expression of ATXN2. The cell clones with homozygous long-repeat alleles, which are rarely obtained from patients with SCA2, showed more drastic reduction of ATXN2 expression than the heterozygous clones. This study thus demonstrates the potential of UKiS, which is a beneficial platform for the efficient development of cell models not only for polyQ diseases but also for any other genetic diseases, which may accelerate our deeper understanding of disease mechanisms and cell-based screening for therapeutic drugs.
Topics: Humans; Glutamine; Peptides; Spinocerebellar Ataxias; Proteins
PubMed: 37821389
DOI: 10.1266/ggs.22-00030 -
Proceedings of the National Academy of... Oct 2023Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many...
Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many disorders including cardiac arrhythmias. Prior work revealed association of mRNAs encoding cardiac Na1.5 () and hERG1 (), but the functional significance of this association was not established. Here, we provide a more comprehensive picture of , , , and transcripts collectively copurifying with nascent hERG1, Na1.5, Ca1.2, or KCNQ1 channel proteins. Single-molecule fluorescence in situ hybridization (smFISH) combined with immunofluorescence reveals that the channel proteins are synthesized predominantly as heterotypic pairs from discrete molecules of mRNA, not as larger cotranslational complexes. Puromycin disrupted colocalization of mRNA with its encoded protein, as expected, but remarkably also pairwise mRNA association, suggesting that transcript association relies on intact translational machinery or the presence of the nascent protein. Targeted depletion of by specific shRNA resulted in concomitant reduction of all associated mRNAs, with a corresponding reduction in the encoded channel currents. This co-knockdown effect, originally described for and , thus appears to be a general phenomenon among transcripts encoding functionally related proteins. In multielectrode array recordings, proarrhythmic behavior arose when I was reduced by the selective blocker dofetilide at IC concentrations, but not when equivalent reductions were mediated by shRNA, suggesting that co-knockdown mitigates proarrhythmic behavior expected from the selective reduction of a single channel species. We propose that coordinated, cotranslational association of functionally related ion channel mRNAs confers electrical stability by co-regulating complementary ion channels in macromolecular complexes.
Topics: Humans; KCNQ1 Potassium Channel; ERG1 Potassium Channel; In Situ Hybridization, Fluorescence; Arrhythmias, Cardiac; RNA, Messenger; RNA, Small Interfering; NAV1.5 Voltage-Gated Sodium Channel
PubMed: 37816059
DOI: 10.1073/pnas.2305295120