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Correction to: I Nature Communications i https://doi.org/10.1038/s41467-022-32639-9, published online 05 September 2022 The original version of this Article contained errors in Fig. 2 is: Graph which replaces the previous incorrect version: Graph This has been corrected in both the PDF and HTML versions of the Article. [Extracted from the article]

The hypoxia-inducible factors (HIFs) regulate the main transcriptional pathway of response to hypoxia in T cells and are negatively regulated by von Hippel-Lindau factor (VHL). But the role of HIFs in the regulation of CD4 T cell responses during infection with M. tuberculosis isn't well understood. Here we show that mice lacking VHL in T cells (Vhl cKO) are highly susceptible to infection with M. tuberculosis, which is associated with a low accumulation of mycobacteria-specific T cells in the lungs that display reduced proliferation, altered differentiation and enhanced expression of inhibitory receptors. In contrast, HIF-1 deficiency in T cells is redundant for M. tuberculosis control. Vhl cKO mice also show reduced responses to vaccination. Further, VHL promotes proper MYC-activation, cell-growth responses, DNA synthesis, proliferation and survival of CD4 T cells after TCR activation. The VHL-deficient T cell responses are rescued by the loss of HIF-1α, indicating that the increased susceptibility to M. tuberculosis infection and the impaired responses of Vhl-deficient T cells are HIF-1-dependent. The role of hypoxia inducible factors in infection and immune response is unclear. Here, the authors study their impact on the regulation of T cells responses during Mycobacteria tuberculosis infection using transcriptomics, flow cytometry and in vivo infection. [ABSTRACT FROM AUTHOR]

Summary: Background The MYC oncogene is one of the most frequently altered driver genes in cancer. MYC is thus a potential target for cancer treatment as well as a biomarker for the disease. However, as a target for treatment, MYC has traditionally been regarded as "undruggable" or difficult to target. We set out to evaluate the efficacy of a novel MYC inhibitor known as MYCMI-6, which acts by preventing MYC from interacting with its cognate partner MAX. Methods MYCMI-6 response was assessed in a panel of breast cancer cell lines using MTT assays and flow cytometry. MYC gene amplification, mRNA and protein expression was analysed using the TCGA and METABRIC databases. Results MYCMI-6 inhibited cell growth in breast cancer cell lines with IC50 values varying form 0.3 μM to >10 μM. Consistent with its ability to decrease cell growth, MYCMI-6 was found to induce apoptosis in two cell lines in which growth was inhibited but not in two cell lines that were resistant to growth inhibition. Across all breast cancers, MYC was found to be amplified in 15.3% of cases in the TCGA database and 26% in the METABRIC database. Following classification of the breast cancers by their molecular subtypes, MYC was most frequently amplified and exhibited highest expression at both mRNA and protein level in the basal subtype. Conclusions Based on these findings, we conclude that for patients with breast cancer, anti-MYC therapy is likely to be most efficacious in patients with the basal subtype. [ABSTRACT FROM AUTHOR]

Activated oncogenes induce compensatory tumour-suppressive responses, such as cellular senescence or apoptosis, but the signals determining the main outcome remain to be fully understood. Here, we uncover a role for Cdk2 (cyclin-dependent kinase 2) in suppressing Myc-induced senescence. Short-term activation of Myc promoted cell-cycle progression in either wild-type or Cdk2 knockout mouse embryo fibroblasts (MEFs). In the knockout MEFs, however, the initial hyper-proliferative response was followed by cellular senescence. Loss of Cdk2 also caused sensitization to Myc-induced senescence in pancreatic β-cells or splenic B-cells in vivo, correlating with delayed lymphoma onset in the latter. Cdk2−/− MEFs also senesced upon ectopic Wnt signalling or, without an oncogene, upon oxygen-induced culture shock. Myc also causes senescence in cells lacking the DNA repair protein Wrn. However, unlike loss of Wrn, loss of Cdk2 did not enhance Myc-induced replication stress, implying that these proteins suppress senescence through different routes. In MEFs, Myc-induced senescence was genetically dependent on the ARF–p53–p21Cip1 and p16INK4a–pRb pathways, p21Cip1 and p16INK4a being selectively induced in Cdk2−/− cells. Thus, although redundant for cell-cycle progression and development, Cdk2 has a unique role in suppressing oncogene- and/or stress-induced senescence. Pharmacological inhibition of Cdk2 induced Myc-dependent senescence in various cell types, including a p53-null human cancer cell line. Our data warrant re-assessment of Cdk2 as a therapeutic target in Myc- or Wnt-driven tumours. [ABSTRACT FROM AUTHOR]

Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes—oligonucleotides attached to antibodies against the two target proteins—guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-γ (IFN-γ) signaling and low-molecular-weight inhibitors. [ABSTRACT FROM AUTHOR]

The c-Myc oncoprotein regulates transcription of genes that are associated with cell growth, proliferation and apoptosis. c-Myc levels are modulated by ubiquitin/proteasome-mediated degradation. Proteasome inhibition leads to c-Myc accumulation within nucleoli, indicating that c-Myc might have a nucleolar function. Here we show that the proteins c-Myc and Max interact in nucleoli and are associated with ribosomal DNA. This association is increased upon activation of quiescent cells and is followed by recruitment of the Myc cofactor TRRAP, enhanced histone acetylation, recruitment of RNA polymerase I (Pol I), and activation of rDNA transcription. Using small interfering RNAs (siRNAs) against c-Myc and an inhibitor of Myc-Max interactions, we demonstrate that c-Myc is required for activating rDNA transcription in response to mitogenic signals. Furthermore, using the ligand-activated MycER (ER, oestrogen receptor) system, we show that c-Myc can activate Pol I transcription in the absence of Pol II transcription. These results suggest that c-Myc coordinates the activity of all three nuclear RNA polymerases, and thereby plays a key role in regulating ribosome biogenesis and cell growth. [ABSTRACT FROM AUTHOR]

Rationale. Recently a new 5-hydroxytryptamine[sub 1B] (5-HT[sub 1B])-receptor antagonist, AR-A000002, was described. It was shown that this compound dose dependently increased 5-HT metabolism and release in guinea pig brain following a single injection. Objectives. The present study investigated effects of 3 weeks twice-daily treatment of guinea pigs with the 5-HT[sub 1B]-receptor antagonist AR-A000002 on the serotonergic neurons and receptor densities. Methods. Guinea pigs were injected subcutaneously with AR-A000002, citalopram or saline twice daily for 3 weeks. Groups of animals were treated with challenge doses of AR-A000002 or saline 24 h after the last chronic treatment (citalopram group 48 h) and sacrificed 2 h thereafter. The effect on 5-HT metabolism and 5-HT release was assessed. Plasma and brain concentrations of AR-A000002 were analysed. The effects on binding of [[sup 3]H]8-OH-DPAT to 5-HT[sub 1A] receptors, [[sup 3]H]GR125743 to 5-HT[sub 1B/1D] receptors, [[sup 3]H]ketanserin to 5-HT[sub 2A] receptors, and [[sup 3]H]prazosin to α[sub 1]-adrenoceptors were determined. Results. Repeated treatment of guinea pigs with AR-A000002 did not change the 5-HT[sub 1B/1D], 5-HT[sub 1A], 5-HT[sub 2A] or α[sub 1]-adrenergic receptor densities. Following repeated treatment of guinea pigs for 3 weeks with AR-A000002, the 5-HT[sub 1B] receptors were still receptive to a challenge with the same compound. Thus, an increase in the 5-HIAA/5-HT ratio and 5-HT release was seen following challenge doses of AR-A000002. No difference in the plasma and brain concentrations of AR-A000002 was found between the sub-chronic treated AR-A000002 and saline-treated guinea pigs. Conclusions. It is concluded that AR-A000002 is a 5-HT[sub 1B] receptor antagonist, which enhances persistently the serotonergic neurotransmission in guinea pig brain. [ABSTRACT FROM AUTHOR]

The terminal 5-HT1B autoreceptors have attracted great pharmacological interest since they are potential targets for compounds modifying serotonergic neurotransmission. In the present work the in vivo biochemical properties of AR-A000002 ((R)-N-[5-methyl-8-(4-methylpiperazin-1-yl)-1,2,3,4-tetrahydro-2-naphthyl]-4-morpholinobenzamide), a novel selective 5-HT1B receptor antagonist, are reported. The effects of AR-A000002 on: 5-HT metabolism was measured as the ratio between 5-HIAA and 5-HT concentrations in different brain regions; 5-HT synthesis was measured as the accumulation of 5-HTP after inhibition of the aromatic amino acid decarboxylase activity with m-hydroxybenzylhydrazine (NSD1015); 5-HT release was measured using the microdialysis technique. 5-HT, 5-HIAA and 5-HTP concentrations were analyzed using high power liquid chromatography (HPLC) with electrochemical detection. AR-A000002 significantly enhanced 5-HT metabolism (5-HIAA/5-HT ratio) and 5-HT synthesis in guinea pig brain in the dose range 0.9–18 mg/kg s.c. (ED50=1 mg/kg s.c. in the four brain regions examined) with maximal effect seen after 2–4 h. AR-A000002 (9 mg/kg s.c.) significantly increased the extracellular concentrations of 5-HT and 5-HIAA by 20% in the guinea pig frontal cortex, measured with the in vivo microdialysis technique in freely moving guinea pigs. AR-A000002 (9 mg/kg s.c.) in combination with the 5-HT uptake inhibitor citalopram (5 mg/kg s.c.) increased the extracellular 5-HT concentration in guinea pig frontal cortex from 250 to 400% of the basal level. Citalopram alone decreased the extracellular 5-HIAA levels to 70% of the basal value. AR-A000002 counteracted the citalopram-induced decrease in 5-HIAA. Since the basal level of extracellular 5-HIAA was 160 times higher than that of 5-HT the 20% increase in 5-HIAA concentrations indicates that only a few percent of the exocytotically released 5-HT from the nerve terminals reached the extracellular space when the re-uptake mechanism was intact. The results also show that the terminal 5-HT1B autoreceptors are tonically activated under drug-free as well as citalopram conditions. The increase in plasma level of cortisol after AR-A000002 administration may indicate stimulation of post-synaptic 5-HT receptors. AR-A000002 also blocked 5-HT1B agonist-induced (CP-135,807) decrease in 5-HT metabolism and hypothermia (ED50=1 mg/kg s.c.), thus indicating competition between these two drugs. It is concluded that AR-A000002 is a 5-HT1B receptor antagonist that enhances the serotonergic neurotransmission in guinea pig brain. [ABSTRACT FROM AUTHOR]

Inhibition of cellular differentiation is one of the well-known biological activities of c-Myc-family proteins. We show here that Myc represses differentiation-induced expression of the cyclin-dependent kinase (CDK) inhibitor p21CIP1 (CDKN1A, p21), known to play an important role in cell fate decisions during growth and differentiation, in hematopoietic cells. Our results demonstrate that the c-Myc-responsive region is situated in the p21 core promoter. c-Myc binds to this region in vitro and in vivo through interaction with the initiator-binding Zn-finger transcription factor Miz-1, which associates directly with the promoter. Association of Myc with the promoter in vivo correlates inversely with p21 expression. Using mutants of c-Myc with impaired binding to Miz-1, our results further show that repression of p21 promoter/reporters as well as the endogenous p21 gene by Myc depends on interaction with Miz-1. Expression of Miz-1 increases during hematopoietic differentiation and Miz-1 activates the p21 promoter under conditions of low Myc levels, indicating a positive role for free Miz-1 in this process. In conclusion, repression of differentia-tion-induced p21 expression through Miz-1 may be an important mechanism by which Myc blocks diffe-rentiation.Oncogene (2003) 22, 351–360. doi:10.1038/sj.onc.1206145 [ABSTRACT FROM AUTHOR]

A large body of evidence has been accumulated that demonstrates dominant effects of Myc proto-oncoproteins on different aspects of cellular growth. Myc is one of the few proteins that is sufficient to drive resting cells into the cell cycle and promote DNA synthesis. In line with this finding is that the constitutive expression of Myc in cells blocks their differentiation. These growth stimulating properties are most likely responsible for Myc's ability to initiate and promote tumor formation. Interestingly Myc can also sensitize cells to apoptosis, suggesting that this protein is part of a life-and-death switch. Molecularly Myc functions as a transcriptional regulator that needs to heterodimerize with Max to exert the biological activities described above and to regulate gene transcription. Myc and Max are just two members of a growing family of proteins referred to as the Myc/Max/Mad network. A hallmark of these proteins is that they possess a C-terminal basic region/helix – loop – helix/leucine zipper domain (bHLHZip). The bHLHZip domain specifies dimerization within the network and determines sequence specific DNA binding. Importantly this domain together with the N-terminal transactivation domain is essential for Myc biology. Here we have summarized the structural, functional, and regulatory aspects of the bHLHZip domain of Myc proteins. [ABSTRACT FROM AUTHOR]

The bHLHZip protein Max interacts with both the Myc and Mad family proteins forming heterodimers which specifically bind certain E-box DNA recognition sequences, thereby regulating transcription. Whereas Myc proteins actively promote cell proliferation, Mad complexes have the opposite function. Although the main regulation of this network seems to be the control of myc- and mad family gene expression, regulation at the level of DNA-binding and transactivation may also be in operation. Few studies on the DNA-binding activity of native Myc : Max or Max : Mad complexes have been reported mainly due to technical difficulties. To overcome these problems we have developed a specific and sensitive solid phase DNA-binding assay based on partial purification of native Myc, Max and Mad1 complexes by immunological methods. Using this technique we report that the DNA-binding activity of c-Myc-containing complexes is reduced during induced differentiation of U-937 monoblasts and F9 embryonic teratocarcinoma cells. In contrast, the DNA-binding of Mad1-containing complexes increases during monocytic differentiation. In general, the DNA-binding activity of c-Myc and Mad1 correlate with their expression. However, our studies of early kinetics of TPA-induced differentiation of U-937 cells as well as of late events during F9 differentiation suggest that post-translational regulation of Myc and Max DNA-binding may also occur. The solid phase DNA-binding assay may thus provide a tool to study the regulation of DNA-binding in more detail. [ABSTRACT FROM AUTHOR]

The aim of the present study was to further investigate the behavioural and biochemical pharmacology of the directly acting dopamine (DA) receptor agonist bromocriptine (BRC). BRC produced an initial depression of locomotion followed after about an hour by a weak but significant locomotor stimulation. The stimulation was potentiated by concomitant administration of the D agonist SKF38393. Ex vivo biochemical determinations indicated that reductions in dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels occurred in the striatum after BRC injection without a significant change in DA levels, indicating a reduced DA turnover. An increase in 5-hydroxytryptamine (5HT) and 5-hydroxyindoleacetic acid (5HIAA) levels occurred in the striatum leading to a significant increase in turnover (i.e. ratio of 5HIAA to 5HT). Noradrenaline concentrations increased in the striatum. In the cortex, sharp falls in HVA and DOPAC levels without a corresponding change in DA were observed. While there was no significant change in noradrenaline levels in this brain region, an increase in 5HIAA, but not in 5HT, levels occurred. These changes indicate an increase in 5HT turnover (ratio of 5HIAA to 5HT). In vivo dialysis indicated that extracellular levels of DA, DOPAC and HVA in the striata of freely moving rats were sharply reduced for at least 6 h after injection. In vitro binding studies showed that BRC exhibited high (K values in low nanomolar range) affinities for DA D, D, D, α and α adrenergic receptors together with unexpectedly high affinity (about 1 nM) for 5HT receptors. The data indicate that the initial behavioural depression and later locomotor stimulation induced by BRC are accompanied by a sharp monophasic fall in striatal extracellular DA levels as indicated by dialysis studies. Since the behavioural stimulation was augmented by concomitant D receptor stimulation, the data suggest that the reduced DA turnover is influencing the amount of DA available to stimulate postsynaptic D receptors. However, the biochemical studies indicated that BRC has a high affinity for 5HT receptors and affects the turnover of 5HT in the brain. Thus, the behavioural effects of BRC may depend not only on effects on the DA system but also on 5HT systems.[/p] [ABSTRACT FROM AUTHOR]

Bromocriptine (BRC) produced a biphasic behavioural effect in mice; an early depressant phase which lasted for about 1 h and a later stimulant phase which lasted from about 1 to 5 h. The stimulation was blocked with SCH23390. Both phases of activity were accompanied by marked striatal DA autoreceptor effects as indicated by reductions in dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels and by a reduction in the accumulation of DOPA (after inhibition of nigrostriatal DA nerve firing and DOPA decarboxylase). However, while the autoreceptor effects were still evident during the behavioural stimulant phase, there was a gradual rise in DOPAC and HVA from 1 to 4 h after injection, indicating a gradually increasing DA turnover. We were unable, using a variety of behavioural and biochemical paradigms, to demonstrate any change in DA autoreceptor sensitivity after one dose of BRC. In electrophysiological studies, however, it was found that prior exposure of rats to one dose of BRC rendered them subsensitive to the rate-inhibiting effects of a second dose of BRC, as measured in anaesthetized animals using extracellular single cell recordings of identified DA neurons in the substantia nigra pars compacta. It is concluded firstly, that the stimulant phase of BRC in mice occurs despite continued occupation of the DA autoreceptors by BRC because adequate endogenous DA is available to provide the required D1 receptor stimulation and secondly, that the terminal autoreceptors in the striatum (as assessed in mice using biochemical techniques) may be regulated differently to the somatodendritic autoreceptors (as assessed electrophysiologically in rats). [ABSTRACT FROM AUTHOR]

The dopamine (DA) D-2 receptor agonists quinpirole (threshold dose, 0.01 mg/kg IP), pergolide (0.025 mg/kg), B-HT 920 (0.003 mg/kg) and (−)-3-PPP (4 mg/kg) produced dose-dependent locomotor depression (immobility) in mice as assessed by a subjective scoring system, with the immobility being characterized by a frozen posture. The animals were still but had their eyes open. The immobility was accompanied by reductions in sniffing, rearing and grooming. The depression (and the associated reduction in the various behaviours) produced by quinpirole (0.1 mg/kg), pergolide (0.1 mg/kg) and B-HT 920 (0.1 mg/kg) was substantially (but not always completely) reversed by the selective D-1 receptor agonist SKF38393 (up to 12 mg/kg) and the non-selective D-1 receptor agonist CY208243 (up to 3 mg/kg). The immobility induced by (−)-3-PPP (16 mg/kg) was also reversed by CY208243 and SKF38393, but the reversal was due to an increase in grooming behaviour in mice challenged with the D-1 receptor agonists, whether or not the animals had also received (−)-3-PPP. There was no reversal of the depression of rearing or sniffing. In contrast, CY208243 and SKF38393 also antagonized the immobility induced by B-HT 920, but the reversal was accompanied by at least partial reversals of the depression of sniffing, rearing and grooming. The reversal of quinpirole-induced immobility by SKF38393 and CY208243 was antagonized by SCH23390 (0.1 mg/kg). The selective D-2 receptor antagonist raclopride (0.025 to 0.4 mg/kg) could not reverse quinpirole-induced immobility. High doses of either raclopride (0.4 mg/kg) or SCH23390 (> 0.1 mg/kg) significantly increased immobility. Although raclopride itself (0.2 mg/kg) produced a substantial increase in DOPAC and homovanillic acid (HVA) levels in the striatum, it did not antagonize the autoreceptor mediated effects of quinpirole (0.1 mg/kg) in reducing the striatal dihydroxyphenylacetic acid (DOPAC) to DA ratio. However, the same dose of raclopride was partly effective in reducing the effects of lower doses of quinpirole (0.01 and 0.03 mg/kg) on the striatal DOPAC to DA ratio. Raclopride (0.2 mg/kg) also partially but significantly reduced the locomotor stimulant effects of d-amphetamine in reserpinized mice. Biochemical analyses in the striata indicated that CY208243 slightly retarded DA turnover (as assessed by the DOPAC/DA ratio). SKF38393 itself also slightly reduced DA turnover. In automated activity cages, using mice depleted of DA with reserpine and a-methyltyrosine, all the D-2 receptor agonists tested, in combination with SKF38393, produced an increase in activity. It is concluded that the depression induced by D-2 receptor agonists is due substantially to a deprivation of DA at postsynaptic D-1 receptors and that, at the doses tested, the D-2 receptor agonists were able to exert measurable stimulation of the postsynaptic receptors. The ability of D-1 receptor agonists to reverse the D-2-mediated depression is due to the stimulation of the postsynaptic D-1 receptors with the injected D-1 receptor agonist (replacing the endogenous transmitter) and the stimulation of the postsynaptic D-2 receptors by the injected D-2 receptor agonist. [ABSTRACT FROM AUTHOR]

The effects on 5-HT turnover (5-HIAA/5-HT ratio) and extracellular 5-HT and 5-HIAA levels (in vivo microdialysis in freely moving animals) were analysed in guinea-pig brains following the 5-HT1B receptor antagonist, GR 127935 { N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-yl) [1,1-biphenyl]-4-carboxamide}, or the 5-HT1A receptor antagonist, WAY-100635 ( N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}- N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride), administered alone or in combination. GR 127935, injected alone, increased 5-HT turnover with maximal effects approximately 50% above the control levels in the four brain regions examined (hypothalamus, hippocampus, striatum and frontal cortex). GR 127935 significantly increased extracellular concentrations of 5-HT and 5-HIAA in frontal cortex (40%), whereas 5-HIAA, but not 5-HT, was elevated in striatum (20–30%). WAY-100635 did not significantly change 5-HT turnover but caused a small significant increase in the extracellular 5-HT and 5-HIAA concentrations in both striatum and frontal cortex. The combined treatment with GR 127935 and WAY-100635 resulted in an increased 5-HT turnover reaching maximal effects of 70–90% above the control values in all brain regions tested and produced a significant elevation of striatal and frontal cortex extracellular 5-HT (40% and 60%, respectively) and 5-HIAA (60% and 70%, respectively) concentrations. The synergistic effect of the two receptor antagonists on the 5-HT turnover and the terminal release of 5-HT indicate somatodendritic 5-HT release and stimulation of inhibitory 5-HT1A receptors at this level. Extracellular 5-HIAA seems to be a better marker than 5-HT itself for the evoked 5-HT release when the reuptake mechanism is intact. [ABSTRACT FROM AUTHOR]