NUDT15 is a key genetic factor for prediction of hematotoxicity in pediatric patients who received a standard low dosage regimen of 6-mercaptopurine

6-Mercaptopurine (6-MP) is commonly used for treatment of acute lymphoblastic leukemia (ALL). The incidence of hematotoxicity caused by this drug is quite high in Asians even using a standard low dosage regimen. The present study was aimed to elucidate the impact of thiopurine S-methyltransferase (TPMT), a nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15), inosine triphosphatase (ITPA) and ATP Binding Cassette Subfamily C Member 4 (ABCC4) polymorphisms on hematotoxicity in pediatric patients who received a standard low starting dose of 6-MP. One hundred and sixty-nine pediatric patients were enrolled and their genotypes were determined. Patients who carried NUDT15¹3 and NUDT15¹2 genotypes were at a 10–15 fold higher risk of severe neutropenia than those of the wild-type during the early months of the maintenance phase. Risk of neutropenia was not significantly increased in patients with other NUDT15 variants as well as in patients with TPMT, ITPA or ABCC4 variants. These results suggest that NUDT15 polymorphisms particularly, NUDT15¹3 and NUDT15¹2, play major roles in 6-MP-induced severe hematotoxicity even when using a standard low dosage of 6-MP and genotyping of these variants is necessary in order to obtain precise tolerance doses and avoid severe hematotoxicity in pediatric patients.     

Insights into the pathogenesis of ATP1A1‐related CMT disease using patient‐specific iPSCs

The development of patient‐specific induced pluripotent stem cells (iPSCs) offered interesting insights in modeling the pathogenesis of Charcot‐Marie‐Tooth (CMT) disease and thus we decided to explore the phenotypes of iPSCs derived from a single CMT patient carrying a mutant ATP1A1 allele (p.Pro600Ala). iPSCs clones generated from CMT and control fibroblasts, were induced to differentiate into neural precursors and then into post‐mitotic neurons. Control iPSCs differentiated into neuronal precursors and then into post‐mitotic neurons within 6‐8 days. On the contrary, the differentiation of CMT iPSCs was clearly defective. Electrophysiological properties confirmed that post‐mitotic neurons were less mature compared to the normal counterpart. The impairment of in vitro differentiation of CMT iPSCs only concerned with the neuronal pathway, because they were able to differentiate into mesendodermal cells and other ectodermal derivatives. ATP1A1 was undetectable in the few neuronal cells derived from CMT iPSCs. ATP1A1 gene mutation (p.Pro600Ala), responsible for a form of axonal CMT disease, is associated in vitro with a dramatic alteration of the differentiation of patient‐derived iPSCs into post‐mitotic neurons. Thus, the defect in neuronal cell development might lead in vivo to a decreased number of mature neurons in ATP1A1‐CMT disease.     

ATP6L promotes metastasis of colorectal cancer by inducing epithelial‐mesenchymal transition

ATP6L, the C subunit of the V‐ATPase V0 domain, is involved in regulating the acidic tumor micro‐environment and may promote tumor progression. However, the expression and functional role of ATP6L in tumors have not yet been well explored. In this study, we found that ATP6L protein overexpression was related to colorectal cancer histological differentiation (P < 0.001), presence of metastasis (P < 0.001) and recurrence (P = 0.02). ATP6L expression in the liver metastatic foci was higher than in the primary foci (P = 0.04). ATP6L expression was notably concomitant with epithelial‐mesenchymal transition (EMT) immunohistochemical features, such as reduced expression of the epithelial marker E‐cadherin (P = 0.021) and increased expression of the mesenchymal marker vimentin (P = 0.004). Results of in vitro and in vivo experiments showed that ATP6L expression could alter cell morphology, regulate EMT‐associated protein expression, and enhance migration and invasion. The effect of ATP6L on metastasis was further demonstrated in a tail vein injection mice model. In addition, the mouse xenograft model showed that ATP6L‐overexpressing HCT116 cells grew into larger tumor masses, showed less necrosis and formed more micro‐vessels than the control cells. Taken together, our results suggest that ATP6L promotes metastasis of colorectal cancer by inducing EMT and angiogenesis, and is a potential target for tumor therapy.     

Adenylate cyclase activator forskolin alleviates intracerebroventricular propionic acid-induced mitochondrial dysfunction of autistic rats

Neuronal mitochondrial dysfunction increases inflammatory mediators and leads to free radical generation and anti-oxidant enzymatic alterations,which are major neuropathological hallmarks responsible for autism.Mitochondrial dysfunction in autism is associated with decreased ATP levels due to reduced levels of cyclic adenosine monophosphate.Rat models of autism were established by intracerebroventricular injection of propionic acid.These rat models had memory dysfunction,decreased muscle coordination and gait imbalance.Biochemical estimation of propionic acid-treated rats showed changes in enzyme activity in neuronal mitochondrial electron transport chain complexes and increases in pro-inflammatory cytokines,oxidative stress and lipid biomarkers.Oral administration of 10,20 and 30 mg/kg adenylate cyclase activator forskolin for 15 days reversed these changes in a dose-dependent manner.These findings suggest that forskolin can alleviate neuronal mitochondrial dysfunction and improve neurological symptoms of rats with autism.This study was approved by the RITS/IAEC,SIRSA,HARYANA on March 3,2014(approval No.RITS/IAEC/2014/03/03).     

Anticonvulsant effect of melatonin through ATP-sensitive channels in mice

Melatonin is a neurohormone secreted principally by the pineal gland. This molecule has various pharmacological properties including improving immune system, prevent cancer, anti-aging, and anti-oxidant effects. The anticonvulsant effects of melatonin have been proved by previous studies. Adenosine triphosphate (ATP)-sensitive potassium (K_ATP) channels are considered as an important target in the seizure modulation. The aim of the present study was to investigate the anticonvulsant effect of melatonin in pentylenetetrazole (PTZ)-induced seizures in mice, focusing on its ability to regulate K_ATP channels. Acute intraperitoneal administration of melatonin (40 and 80 mg/kg) increased clonic seizure threshold induced by intravenous administration of PTZ. Melatonin (40 and 80 mg/kg) increased the latency of clonic seizure and reduced its frequency in mice receiving an intraperitoneal injection of PTZ. Administration of glibenclamide, a K_ATP channels blocker, before intravenous injection of PTZ reduced melatonin anticonvulsant effect. Diazoxide and cromakalim, as K_ATP channels openers, increased antiseizure effect of melatonin in PTZ model of seizures. These findings suggest that the antiseizure effect of melatonin probably is gained through increasing the opening of K_ATP channels.     

P2Y13 and P2X7 receptors modulate mechanically induced adenosine triphosphate release from mast cells

Subcutaneous mast cells (MCs) are vulnerable to mechanical stimulation from external environment. Thus, MCs immune function could be modulated by their mechanosensitivity. This property has been identified as the trigger mechanism of needling acupuncture, a traditional oriental therapy. Previously we have demonstrated the release of adenosine triphosphate (ATP), a stress-responsive signalling molecule, from mechanical-perturbed MCs. The current work explores its underlying mechanisms. We noticed that propagation of intracellular free Ca²⁺ occurred among HMC-1 cells in response to 50% hypotonic shock. Additionally, amplifying cascade of ATP-induced ATP release was observed in RBL-2H3 cells stimulated by medium displacement, which could be mimicked by exogenous ATP (exoATP). Secondary ATP liberation induced by low level (50 nmol/L) of exoATP was reduced by inhibiting ecto-ATPase-dependent ADP production with ARL67156, or blocking P2 receptors with suramin or PPADS, or with specific P2Y₁₃ receptor antagonist MRS2211, or siRNA. Secondary ATP release induced by higher dose (200 μmol/L) of exoATP, sufficient to stimulate P2X₇ receptor, was attenuated by suramin, PPADS or specific P2X₇ receptor antagonist BBG, or siRNA. Finally, RT-PCR confirmed mRNA expression of P2Y₁₃ and P2X₇ in RBL-2H3 cells. Additionally, such secondary ATP release was attenuated by DPCPX, specific antagonist of adenosine A1 receptor, but not by MRS2179, specific inhibitor of P2Y₁ receptor. In summary, mechanosensitive ATP release from MCs is facilitated by paracrine/autocrine stimulation of P2Y₁₃ and P2X₇ receptors. This multi-receptor combination could mediate transmission of information from a local site to distal areas, enabling communication with multiple surrounding cells to coordinate and synchronize their function.     

Cutaneous nociception: Role of keratinocytes

Recent years have brought an enhanced understanding of keratinocyte contribution to cutaneous nociception. While intra‐epidermal nerve endings were classically considered as the exclusive transducers of cutaneous noxious stimuli, it has now been demonstrated that epidermal keratinocytes can initiate nociceptive responses, like Merkel cells do for the innocuous mechanotransduction. In the light of recent in vivo findings, this article outlines this paradigm shift that points to a not yet considered population of sensory epidermal cells.     

The Mineralization Regulator ANKH Mediates Cellular Efflux of ATP,Not Pyrophosphate

The plasma membrane protein ankylosis homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints. We recently showed that when overproduced in HEK293 cells, ANKH mediates release of large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. ATP is converted extracellularly into PPi and AMP by the ectoenzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANKH. We now addressed the question of whether PPi leaves cells via ANKH using HEK293 cells that completely lack ENPP1. Introduction of ANKH in these ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi found in ENPP1-proficient cells. Ank activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1^−/− mice contained <2.5% of the PPi found in bones of wild-type mice, showing that Enpp1 activity is also a prerequisite for Ank-dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1-mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60% to 70% of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification therefore do so by extruding NTPs rather than PPi itself. © 2022 American Society for Bone and Mineral Research (ASBMR).