Detection of Novel t(12;17)(p12;p13) in Relapsed Refractory Acute Myeloid Leukemia by Anchored Multiplex PCR(AMP)-based Next-Generation Sequencing.

Although several technologies can be used to detect gene fusions, anchored multiplex PCR next-generation sequencing (AMP-NGS) offers the advantage of novel fusion detection and the ability to multiplex multitudinous genes. We applied AMP-NGS technology in the evaluation of a 56-year-old gentleman with myelodysplastic syndrome transformed acute myeloid leukemia (AML). Patient was initially diagnosed with low-risk myelodysplastic syndrome-refractory cytopenias and multilineage dysplasia (MDS-RCMD), progressed to AML after failing hypomethylating agent therapy. At progression patients had normal cytogenetics but NGS profiling showed ETV6 c.416_417del CT frame shift and U2AF1 S34F mutations. Patient attains brief remission of 2 months after induction chemotherapy and then he was refractory to 2 salvage chemotherapy regimens. Reassessment after failing second salvage, identified t(12;17)(p13;p13)[20] by karyotype. It was postulated that the 12p13 locus might represent a new rearrangement of ETV6. AMP-NGS confirmed involvement of the ETV6 with discovery of a novel fusion partner, HIC1. The detection of the novel fusion partners was supported by the breakpoints originally observed by karyotype. This discovery of ETV6-HIC1 gene fusion by AMP-NGS technology provided new insight into a leukemogenic pathway in AML. Future use of this technology can serve as an adjunct tool in workup of patients with AML and can also help in formulating therapeutic strategies.

Noninvasive Detection of ctDNA Reveals Intratumor Heterogeneity and Is Associated with Tumor Burden in Gastrointestinal Stromal Tumor.

Molecular analysis of circulating tumor DNA (ctDNA) has a large potential for clinical application by capturing tumor-specific aberrations through noninvasive sampling. In gastrointestinal stromal tumor (GIST), analysis of KIT and PDGFRA mutations is important for therapeutic decisions, but the invasiveness of traditional biopsies limits the possibilities for repeated sampling. Using targeted next-generation sequencing, we have analyzed circulating cell-free DNA from 50 GIST patients. Tumor-specific mutations were detected in 16 of 44 plasma samples (36%) from treatment-naïve patients and in three of six (50%) patients treated with tyrosine kinase inhibitors. A significant association between detection of ctDNA and the modified National Institutes of Health risk classification was found. All patients with metastatic disease had detectable ctDNA, and tumor burden was the most important detection determinant. Median tumor size was 13.4 cm for patients with detectable mutation in plasma compared with 4.4 cm in patients without detectable mutation (P = 0.006). ctDNA analysis of a patient with disease progression on imatinib revealed that multiple resistance mutations were synchronously present, and detailed analysis of tumor tissue showed that these were spatially distributed in the primary tumor. Plasma samples taken throughout the course of treatment demonstrated that clonal evolution can be monitored over time. In conclusion, we have shown that detection of GIST-specific mutations in plasma is particularly feasible for patients with high tumor burden. In such cases, we have demonstrated that mutational analysis by use of liquid biopsies can capture the molecular heterogeneity of the whole tumor, and may guide treatment decisions during progression. Mol Cancer Ther; 17(11); 2473-80. ©2018 AACR.

Molecular Profiling of Hyalinizing Clear Cell Carcinomas Revealed a Subset of Tumors Harboring a Novel EWSR1-CREM Fusion: Report of 3 Cases.

We describe a novel gene fusion, EWSR1-CREM, identified in 3 cases of clear cell carcinoma (CCC) using anchored multiplex polymerase chain reaction, a next-generation sequencing-based technique. CCC is a low-grade salivary tumor recently characterized to have EWSR1-ATF1 fusions in the majority of cases. Three cases of malignant tumor presenting in the base of tongue, lung, and nasopharynx were studied. All cases shared a clear cell morphology with hyalinized stroma, presence of mucin and p63 positivity and were initially diagnosed as mucoepidermoid carcinoma but were negative for evidence of any of the expected gene fusions. Anchored multiplex polymerase chain reaction demonstrated a EWSR1-CREM fusion in all 3 cases to confirm a diagnosis of CCC. This finding is biologically justified as CREM and ATF1 both belong to the CREB family of transcription factors. EWSR1-CREM fusions have not been previously reported in CCC and have only rarely been reported in other tumors. We show that the ability to discover novel gene variants with next-generation sequencing-based assays has clinical utility in the pathologic classification of fusion gene-associated tumors.

ALK Is a Specific Diagnostic Marker for Inflammatory Myofibroblastic Tumor of the Uterus.

Inflammatory myofibroblastic tumor (IMT) is a myofibroblastic/fibroblastic neoplasm of intermediate malignant potential. It is frequently characterized by genetic fusion of ALK with a variety of partner genes, which results in the activated ALK signaling pathway that can be targeted with kinase inhibitors. IMTs can occur in the gynecologic tract, with the uterus (corpus and cervix) being the most frequent site. Recent studies suggest that IMTs in the gynecologic tract are underrecognized, and a low-threshold for performing ALK immunohistochemistry has been proposed. The aim of this study was to evaluate the specificity of ALK immunohistochemistry for IMTs among uterine mesenchymal and mixed epithelial/mesenchymal tumors. We performed ALK immunohistochemistry on 14 molecularly confirmed uterine IMTs and 260 other uterine pure mesenchymal and mixed epithelial/mesenchymal tumors. Cases showing any positive cytoplasmic and/or membranous staining of the tumor cells were considered to be ALK positive. All 14 IMTs were confirmed to harbor ALK genetic fusion by RNA sequencing, and ALK immunostaining in the form of granular cytoplasmic positivity with paranuclear accentuation was observed in all 14 cases. ALK was negative (complete absence of staining) in all the other pure mesenchymal tumors and in all mixed epithelial/mesenchymal tumors examined. Our findings show that ALK is a highly specific diagnostic immunohistochemical marker for ALK fusion in uterine mesenchymal tumors. In the work-up of uterine mesenchymal tumors, particularly smooth muscle tumors showing myxoid stromal changes, a diagnosis of IMT should be strongly considered if ALK positivity is observed.

Uterine Inflammatory Myofibroblastic Tumors Frequently Harbor ALK Fusions With IGFBP5 and THBS1.

Inflammatory myofibroblastic tumor (IMT) can occur in a number of anatomic sites, including the uterus. Like its soft tissue counterpart, uterine IMT frequently expresses ALK and harbors ALK genetic rearrangements. The aim of this study is to fully characterize the genetic fusions that occur in uterine IMT. We studied 11 uterine IMTs with typical histology and 8 uterine myxoid smooth muscle tumors (5 leiomyomas, 1 smooth muscle tumor of uncertain malignant potential, and 2 leiomyosarcomas) in which the differential of IMT was considered, using a RNA-sequencing-based fusion assay to detect genetic fusions involving ALK, ROS1, RET, NTRK1/3, and other genes. ALK was expressed in 10 of 11 IMTs and 1 tumor initially categorized as a myxoid leiomyoma (granular cytoplasmic staining with paranuclear accentuation). Fusion transcripts involving ALK were identified in 9 of 10 ALK immunopositive IMTs, with 3 harboring IGFBP5-ALK, 3 harboring THBS1-ALK, 2 harboring FN1-ALK, and 1 harboring TIMP3-ALK. Among the smooth muscle tumors, IGFBP5-ALK fusion transcript was identified in only 1 ALK immunopositive case. Further review revealed that although a diagnosis of IMT was considered for the ALK immunopositive myxoid leiomyoma, this diagnosis was not initially rendered only because fluorescence in situ hybridization analysis was interpreted as negative for ALK genetic rearrangement; this case is best reclassified as an IMT. Notably, all the ALK fusions identified in our study included the transmembrane domain-encoding exon 19 of ALK. Our findings confirm the high frequency of ALK fusions in uterine IMT, with an enrichment of novel 5' ALK fusion partners (IGFBP5, THBS1, and TIMP3) and exon 19-containing ALK fusion. Given that IGFBP5 and FN1 are both situated on the same chromosome as ALK, fluorescence in situ hybridization analysis for ALK rearrangement may not be reliable and a negative result should not exclude a diagnosis of uterine IMT if the histologic features and ALK immunostaining findings are supportive.

The application of next-generation sequencing-based molecular diagnostics in endometrial stromal sarcoma.

AIMS: Endometrial stromal sarcomas (ESSs) are divided into low-grade and high-grade subtypes, with the latter showing more aggressive clinical behaviour. Although histology and immunophenotype can aid in the diagnosis of these tumours, genetic studies can provide additional diagnostic insights, as low-grade ESSs frequently harbour fusions involving JAZF1/SUZ12 and/or JAZF1/PHF1, whereas high-grade ESSs are defined by YWHAE-NUTM2A/B fusions. The aim of this study was to evaluate the utility of a next-generation sequencing (NGS)-based assay in identifying ESS fusions in archival formalin-fixed paraffin-embedded tumour samples. METHODS AND RESULTS: We applied an NGS-based fusion transcript detection assay (Archer FusionPlex Sarcoma Panel) that targets YWHAE and JAZF1 fusions in a series of low-grade ESSs (n = 11) and high-grade ESSs (n = 5) that were previously confirmed to harbour genetic rearrangements by fluorescence in-situ hybridization (FISH) and/or reverse transcription polymerase chain reaction (RT-PCR) analyses. The fusion assay identified junctional fusion transcript sequences that corresponded to the known FISH/RT-PCR results in all cases. Four low-grade ESSs harboured JAZF1-PHF1 fusions with different junctional sequences, and all were correctly identified because of the open-ended nature of the assay design, using anchored multiplex polymerase chain reaction. Seven non-ESS sarcomas were also included as negative controls, and no strong ESS fusion candidates were identified in these cases. CONCLUSIONS: Our findings demonstrate good sensitivity and specificity of an NGS-based gene fusion assay in the detection of ESS fusion transcripts.

Functional analysis of neuronal microRNAs in Caenorhabditis elegans dauer formation by combinational genetics and Neuronal miRISC immunoprecipitation.

Identifying the physiological functions of microRNAs (miRNAs) is often challenging because miRNAs commonly impact gene expression under specific physiological conditions through complex miRNA::mRNA interaction networks and in coordination with other means of gene regulation, such as transcriptional regulation and protein degradation. Such complexity creates difficulties in dissecting miRNA functions through traditional genetic methods using individual miRNA mutations. To investigate the physiological functions of miRNAs in neurons, we combined a genetic "enhancer" approach complemented by biochemical analysis of neuronal miRNA-induced silencing complexes (miRISCs) in C. elegans. Total miRNA function can be compromised by mutating one of the two GW182 proteins (AIN-1), an important component of miRISC. We found that combining an ain-1 mutation with a mutation in unc-3, a neuronal transcription factor, resulted in an inappropriate entrance into the stress-induced, alternative larval stage known as dauer, indicating a role of miRNAs in preventing aberrant dauer formation. Analysis of this genetic interaction suggests that neuronal miRNAs perform such a role partly by regulating endogenous cyclic guanosine monophosphate (cGMP) signaling, potentially influencing two other dauer-regulating pathways. Through tissue-specific immunoprecipitations of miRISC, we identified miRNAs and their likely target mRNAs within neuronal tissue. We verified the biological relevance of several of these miRNAs and found that many miRNAs likely regulate dauer formation through multiple dauer-related targets. Further analysis of target mRNAs suggests potential miRNA involvement in various neuronal processes, but the importance of these miRNA::mRNA interactions remains unclear. Finally, we found that neuronal genes may be more highly regulated by miRNAs than intestinal genes. Overall, our study identifies miRNAs and their targets, and a physiological function of these miRNAs in neurons. It also suggests that compromising other aspects of gene expression, along with miRISC, can be an effective approach to reveal miRNA functions in specific tissues under specific physiological conditions.

Systematic analysis of tissue-restricted miRISCs reveals a broad role for microRNAs in suppressing basal activity of the C. elegans pathogen response.

Gene regulation by microRNAs (miRNAs) under specific physiological conditions often involves complex interactions between multiple miRNAs and a large number of their targets, as well as coordination with other regulatory mechanisms, limiting the effectiveness of classical genetic methods to identify miRNA functions. We took a systematic approach to analyze the miRNA-induced silencing complex (miRISC) in individual tissues of C. elegans and found that mRNAs encoded by pathogen-responsive genes were dramatically overrepresented in the intestinal miRISC, and that multiple miRNAs accumulated in the intestinal miRISCs upon infection. Inactivation of the miRISC or ablation of miRNAs from multiple families resulted in overexpression of several pathogen-responsive genes under basal conditions and, surprisingly, enhanced worm survival on pathogenic Pseudomonas aeruginosa. These results indicate that much of the miRNA activity in the gut is dedicated to attenuating the activity of the pathogen-response system, uncovering a complex physiological function of the miRNA network.

Systematic analysis of dynamic miRNA-target interactions during C. elegans development.

Although microRNA (miRNA)-mediated functions have been implicated in many aspects of animal development, the majority of miRNA::mRNA regulatory interactions remain to be characterized experimentally. We used an AIN/GW182 protein immunoprecipitation approach to systematically analyze miRNA::mRNA interactions during C. elegans development. We characterized the composition of miRNAs in functional miRNA-induced silencing complexes (miRISCs) at each developmental stage and identified three sets of miRNAs with distinct stage-specificity of function. We then identified thousands of miRNA targets in each developmental stage, including a significant portion that is subject to differential miRNA regulation during development. By identifying thousands of miRNA family-mRNA pairs with temporally correlated patterns of AIN-2 association, we gained valuable information on the principles of physiological miRNA::target recognition and predicted 1589 high-confidence miRNA family::mRNA interactions. Our data support the idea that miRNAs preferentially target genes involved in signaling processes and avoid genes with housekeeping functions, and that miRNAs orchestrate temporal developmental programs by coordinately targeting or avoiding genes involved in particular biological functions.

Suppression of proliferative defects associated with processing-defective lamin A mutants by hTERT or inactivation of p53.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, debilitating disease with early mortality and rapid onset of aging-associated pathologies. It is linked to mutations in LMNA, which encodes A-type nuclear lamins. The most frequent HGPS-associated LMNA mutation results in a protein, termed progerin, with an internal 50 amino acid deletion and, unlike normal A-type lamins, stable farnesylation. The cellular consequences of progerin expression underlying the HGPS phenotype remain poorly understood. Here, we stably expressed lamin A mutants, including progerin, in otherwise identical primary human fibroblasts to compare the effects of different mutants on nuclear morphology and cell proliferation. We find that expression of progerin leads to inhibition of proliferation in a high percentage of cells and slightly premature senescence in the population. Expression of a stably farnesylated mutant of lamin A phenocopied the immediate proliferative defects but did not result in premature senescence. Either p53 inhibition or, more surprisingly, expression of the catalytic subunit of telomerase (hTERT) suppressed the early proliferative defects associated with progerin expression. These findings lead us to propose that progerin may interfere with telomere structure or metabolism in a manner suppressible by increased telomerase levels and possibly link mechanisms leading to progeroid phenotypes to those of cell immortalization.

Single-gene deletions that restore mating competence to diploid yeast.

Using the Saccharomyces cerevisiae MATa/MATalpha ORF deletion collection, homozygous deletion strains were identified that undergo mating with MATa or MATalpha haploids. Seven homozygous deletions were identified that confer enhanced mating. Three of these, lacking CTF8, CTF18, and DCC1, mate at a low frequency with either MATa or MATalpha haploids. The products of these genes form a complex involved in sister chromatid cohesion. Each of these strains also exhibits increased chromosome loss rates, and mating likely occurs due to loss of one copy of chromosome III, which bears the MAT locus. Three other homozygous diploid deletion strains, ylr193cDelta/ylr193cDelta, yor305wDelta/yor305wDelta, and ypr170cDelta/ypr170cDelta, mate at very low frequencies with haploids of either or both mating types. However, an ist3Delta/ist3Delta strain mates only with MATa haploids. It is shown that IST3, previously linked to splicing, is required for efficient processing of the MATa1 message, particularly the first intron. As a result, the ist3Delta/ist3Delta strain expresses unbalanced ratios of Matalpha to Mata proteins and therefore mates with MATa haploids. Accordingly, mating in this diploid can be repressed by introduction of a MATa1 cDNA. In summary, this study underscores and elaborates upon predicted pathways by which mutations restore mating function to yeast diploids and identifies new mutants warranting further study.

Werner and Hutchinson-Gilford progeria syndromes: mechanistic basis of human progeroid diseases.

Progeroid syndromes have been the focus of intense research in part because they might provide a window into the pathology of normal ageing. Werner syndrome and Hutchinson-Gilford progeria syndrome are two of the best characterized human progeroid diseases. Mutated genes that are associated with these syndromes have been identified, mouse models of disease have been developed, and molecular studies have implicated decreased cell proliferation and altered DNA-damage responses as common causal mechanisms in the pathogenesis of both diseases.

Aging: progeria and the lamin connection.

The relationship between progerias--diseases that resemble premature aging--and the normal aging process has been a source of debate in the aging research community. A recent study finds that LMNA, a gene targeted for mutation in Hutchinson Gilford Progeria Syndrome, may control the onset of aging-associated decline in normal fibroblasts.

Stabilization of the retinoblastoma protein by A-type nuclear lamins is required for INK4A-mediated cell cycle arrest.

Mutations in the LMNA gene, which encodes all A-type lamins, including lamin A and lamin C, cause a variety of tissue-specific degenerative diseases termed laminopathies. Little is known about the pathogenesis of these disorders. Previous studies have indicated that A-type lamins interact with the retinoblastoma protein (pRB). Here we probe the functional consequences of this association and further examine links between nuclear structure and cell cycle control. Since pRB is required for cell cycle arrest by p16(ink4a), we tested the responsiveness of multiple lamin A/C-depleted cell lines to overexpression of this CDK inhibitor and tumor suppressor. We find that the loss of A-type lamin expression results in marked destabilization of pRB. This reduction in pRB renders cells resistant to p16(ink4a)-mediated G(1) arrest. Reintroduction of lamin A, lamin C, or pRB restores p16(ink4a)-responsiveness to Lmna(-/-) cells. An array of lamin A mutants, representing a variety of pathologies as well as lamin A processing mutants, was introduced into Lmna(-/-) cells. Of these, a mutant associated with mandibuloacral dysplasia (MAD R527H), as well as two lamin A processing mutants, but not other disease-associated mutants, failed to restore p16(ink4a) responsiveness. Although our findings do not rule out links between altered pRB function and laminopathies, they fail to support such an assertion. These findings do link lamin A/C to the functional activation of a critical tumor suppressor pathway and further the possibility that somatic mutations in LMNA contribute to tumor progression.

Lamin A/C and emerin are critical for skeletal muscle satellite cell differentiation.

Mutations within LMNA, encoding A-type nuclear lamins, are associated with multiple tissue-specific diseases, including Emery-Dreifuss (EDMD2/3) and Limb-Girdle muscular dystrophy (LGMD1B). X-linked EDMD results from mutations in emerin, a lamin A-associated protein. The mechanisms through which these mutations cause muscular dystrophy are not understood. Here we show that most, but not all, cultured muscle cells from lamin A/C knockout mice exhibit impaired differentiation kinetics and reduced differentiation potential. Similarly, normal muscle cells that have been RNA interference (RNAi) down-regulated for either A-type lamins or emerin have impaired differentiation potentials. Replicative myoblasts lacking A-type lamins or emerin also have decreased levels of proteins important for muscle differentiation including pRB, MyoD, desmin, and M-cadherin; up-regulated Myf5; but no changes in Pax3, Pax7, MEF2C, MEF2D, c-met, and beta-catenin. To determine whether impaired myogenesis is linked to reduced MyoD or desmin levels, these proteins were individually expressed in Lmna(-/-) myoblasts that were then induced to undergo myogenesis. Expression of either MyoD or, more surprisingly, desmin in Lmna(-/-) myoblasts resulted in increased differentiation potential. These studies indicate roles for A-type lamins and emerin in myogenic differentiation and also suggest that these effects are at least in part due to decreased endogenous levels of other critical myoblast proteins. The delayed differentiation kinetics and decreased differentiation potential of lamin A/C-deficient and emerin-deficient myoblasts may in part underlie the dystrophic phenotypes observed in patients with EDMD.

HIV protease inhibitors block adipocyte differentiation independently of lamin A/C.

OBJECTIVES: To determine the importance of lamin A/C for fat cell differentiation in vitro and for the anti-adipogenic activity of HIV protease inhibitors such as indinavir. METHODS: Lipodystrophy-associated and processing-defective mutants of lamin A were stably expressed at high levels in 3T3-L1 pre-adipocytes. Additionally, 3T3-L1 pre-adipocytes with stable reduction of lamin A/C or emerin were derived. The cells were differentiated for 8 days into mature adipocytes in the presence or absence of indinavir or nelfinavir. RESULTS: 3T3-L1 cells stably expressing high levels of lipodystrophy-associated or processing-defective mutants of lamin A differentiated with comparable efficiencies to control cells. Similarly, cells with dramatically reduced lamin A levels differentiated as efficiently as controls. Although indinavir stimulated the accumulation of unprocessed lamin A, cells with dramatically reduced lamin A/C levels and no detectable prelamin A remained responsive to an indinavir-induced inhibition of adipogenesis. CONCLUSIONS: The ability of HIV protease inhibitor to stimulate the accumulation of unprocessed lamin A is neither necessary nor sufficient to explain their anti-adipogenic activity. Furthermore, lamin A/C plays a minimal role in the differentiation of 3T3-L1.

A-type nuclear lamins, progerias and other degenerative disorders.

Nuclear lamins were identified as core nuclear matrix constituents over 20 years ago. They have been ascribed structural roles such as maintaining nuclear integrity and assisting in nuclear envelope formation after mitosis, and have also been linked to nuclear activities including DNA replication and transcription. Recently, A-type lamin mutations have been linked to a variety of rare human diseases including muscular dystrophy, lipodystrophy, cardiomyopathy, neuropathy and progeroid syndromes (collectively termed laminopathies). Most diseases arise from dominant, missense mutations, leading to speculation as to how different mutations in the same gene can give rise to such a diverse set of diseases, some of which share little phenotypic overlap. Understanding the cellular dysfunctions that lead to laminopathies will almost certainly provide insight into specific roles of A-type lamins in nuclear organization. Here, we compare and contrast the LMNA mutations leading to laminopathies with emphasis on progerias, and discuss possible functional roles for A-type lamins in the maintenance of healthy tissues.

Nuclear reorganization of mammalian DNA synthesis prior to cell cycle exit.

In primary mammalian cells, DNA replication initiates in a small number of perinucleolar, lamin A/C-associated foci. During S-phase progression in proliferating cells, replication foci distribute to hundreds of sites throughout the nucleus. In contrast, we find that the limited perinucleolar replication sites persist throughout S phase as cells prepare to exit the cell cycle in response to contact inhibition, serum starvation, or replicative senescence. Proteins known to be involved in DNA synthesis, such as PCNA and DNA polymerase delta, are concentrated in perinucleolar foci throughout S phase under these conditions. Moreover, chromosomal loci are redirected toward the nucleolus and overlap with the perinucleolar replication foci in cells poised to undergo cell cycle exit. These same loci remain in the periphery of the nucleus during replication under highly proliferative conditions. These results suggest that mammalian cells undergo a large-scale reorganization of chromatin during the rounds of DNA replication that precede cell cycle exit.

LMNA mutations in atypical Werner's syndrome.

BACKGROUND: Werner's syndrome is a progeroid syndrome caused by mutations at the WRN helicase locus. Some features of this disorder are also present in laminopathies caused by mutant LMNA encoding nuclear lamin A/C. Because of this similarity, we sequenced LMNA in individuals with atypical Werner's syndrome (wild-type WRN). METHODS: Of 129 index patients referred to our international registry for molecular diagnosis of Werner's syndrome, 26 (20%) had wildtype WRN coding regions and were categorised as having atypical Werner's syndrome on the basis of molecular criteria. We sequenced all exons of LMNA in these individuals. Mutations were confirmed at the mRNA level by RT-PCR sequencing. In one patient in whom an LMNA mutation was detected and fibroblasts were available, we established nuclear morphology and subnuclear localisation. FINDINGS: In four (15%) of 26 patients with atypical Werner's syndrome, we noted heterozygosity for novel missense mutations in LMNA, specifically A57P, R133L (in two people), and L140R. The mutations altered relatively conserved residues within lamin A/C. Fibroblasts from the patient with the L140R mutation had a substantially enhanced proportion of nuclei with altered morphology and mislocalised lamins. Individuals with atypical Werner's syndrome with mutations in LMNA had a more severe phenotype than did those with the disorder due to mutant WRN. INTERPRETATION: Our findings indicate that Werner's syndrome is molecularly heterogeneous, and a subset of the disorder can be judged a laminopathy.

Extracellular ATP signaling and P2X nucleotide receptors in monolayers of primary human vascular endothelial cells.

ATP and its metabolites regulate vascular tone; however, the sources of the ATP released in vascular beds are ill defined. As such, we tested the hypothesis that all limbs of an extracellular purinergic signaling system are present in vascular endothelial cells: ATP release, ATP receptors, and ATP receptor-triggered signal transduction. Primary cultures of human endothelial cells derived from multiple blood vessels were grown as monolayers and studied using a bioluminescence detection assay for ATP released into the medium. ATP is released constitutively and exclusively across the apical membrane under basal conditions. Hypotonic challenge or the calcium agonists ionomycin and thapsigargin stimulate ATP release in a reversible and regulated manner. To assess expression of P2X purinergic receptor channel subtypes (P2XRs), we performed degenerate RT-PCR, sequencing of the degenerate P2XR product, and immunoblotting with P2XR subtype-specific antibodies. Results revealed that P2X(4) and P2X(5) are expressed abundantly by endothelial cell primary cultures derived from multiple blood vessels. Together, these results suggest that components of an autocrine purinergic signaling loop exist in the endothelial cell microvasculature that may allow for "self-regulation" of endothelial cell function and modulation of vascular tone.