Transcriptomic variation of pharmacogenes in multiple human tissues and lymphoblastoid cell lines.

Variation in the expression level and activity of genes involved in drug disposition and action ('pharmacogenes') can affect drug response and toxicity, especially when in tissues of pharmacological importance. Previous studies have relied primarily on microarrays to understand gene expression differences, or have focused on a single tissue or small number of samples. The goal of this study was to use RNA-sequencing (RNA-seq) to determine the expression levels and alternative splicing of 389 Pharmacogenomics Research Network pharmacogenes across four tissues (liver, kidney, heart and adipose) and lymphoblastoid cell lines, which are used widely in pharmacogenomics studies. Analysis of RNA-seq data from 139 different individuals across the 5 tissues (20-45 individuals per tissue type) revealed substantial variation in both expression levels and splicing across samples and tissue types. Comparison with GTEx data yielded a consistent picture. This in-depth exploration also revealed 183 splicing events in pharmacogenes that were previously not annotated. Overall, this study serves as a rich resource for the research community to inform biomarker and drug discovery and use.

Allelic mRNA expression imbalance in C-type lectins reveals a frequent regulatory SNP in the human surfactant protein A (SP-A) gene.

Genetic variation in C-type lectins influences infectious disease susceptibility but remains poorly understood. We used allelic mRNA expression imbalance (AEI) technology for surfactant protein (SP)-A1, SP-A2, SP-D, dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), macrophage mannose receptor (MRC1) and Dectin-1, expressed in human macrophages and/or lung tissues. Frequent AEI, an indicator of regulatory polymorphisms, was observed in SP-A2, SP-D and DC-SIGN. AEI was measured for SP-A2 in 38 lung tissues using four marker single-nucleotide polymorphisms (SNPs) and was confirmed by next-generation sequencing of one lung RNA sample. Genomic DNA at the SP-A2 DNA locus was sequenced by Ion Torrent technology in 16 samples. Correlation analysis of genotypes with AEI identified a haplotype block, and, specifically, the intronic SNP rs1650232 (30% minor allele frequency); the only variant consistently associated with an approximately twofold change in mRNA allelic expression. Previously shown to alter a NAGNAG splice acceptor site with likely effects on SP-A2 expression, rs1650232 generates an alternative splice variant with three additional bases at the start of exon 3. Validated as a regulatory variant, rs1650232 is in partial linkage disequilibrium with known SP-A2 marker SNPs previously associated with risk for respiratory diseases including tuberculosis. Applying functional DNA variants in clinical association studies, rather than marker SNPs, will advance our understanding of genetic susceptibility to infectious diseases.

Intronic polymorphism in CYP3A4 affects hepatic expression and response to statin drugs.

Cytochrome P450 3A4 (CYP3A4) metabolizes ∼50% of all clinically used drugs. Although CYP3A4 expression varies widely between individuals, the contribution of genetic factors remains uncertain. In this study, we measured allelic CYP3A4 heteronuclear RNA (hnRNA) and mRNA expression in 76 human liver samples heterozygous for at least one of eight marker SNPs and found marked allelic expression imbalance (1.6-6.3-fold) in 10/76 liver samples (13%). This was fully accounted for by an intron 6 SNP (rs35599367, C>T), which also affected mRNA expression in cell culture on minigene transfections. CYP3A4 mRNA level and enzyme activity in livers with CC genotype were 1.7- and 2.5-fold, respectively, greater than in CT and TT carriers. In 235 patients taking stable doses of atorvastatin, simvastatin, or lovastatin for lipid control, carriers of the T allele required significantly lower statin doses (0.2-0.6-fold, P=0.019) than non-T carriers for optimal lipid control. These results indicate that intron 6 SNP rs35599367 markedly affects expression of CYP3A4 and could serve as a biomarker for predicting response to CYP3A4-metabolized drugs.

An opiate binding site in the rat brain is highly selective for 4,5-epoxymorphinans.

In vitro binding studies have demonstrated the existence of multiple opiate receptor types. An additional site in the rat brain (termed the lambda site) is distinct from the established types by its selectivity for 4,5-epoxymorphinans (such as naloxone and morphine). While the lambda site displays a high affinity for naloxone in vivo and in vitro in fresh brain membrane homogenates, these sites rapidly convert in vitro to a state of low affinity. The regional distribution of the lambda site in the brain is strikingly different from that of the classic opiate receptor types.

The Pharmacogenomics Research Network Translational Pharmacogenetics Program: Outcomes and Metrics of Pharmacogenetic Implementations Across Diverse Healthcare Systems.

Numerous pharmacogenetic clinical guidelines and recommendations have been published, but barriers have hindered the clinical implementation of pharmacogenetics. The Translational Pharmacogenetics Program (TPP) of the National Institutes of Health (NIH) Pharmacogenomics Research Network was established in 2011 to catalog and contribute to the development of pharmacogenetic implementations at eight US healthcare systems, with the goal to disseminate real-world solutions for the barriers to clinical pharmacogenetic implementation. The TPP collected and normalized pharmacogenetic implementation metrics through June 2015, including gene-drug pairs implemented, interpretations of alleles and diplotypes, numbers of tests performed and actionable results, and workflow diagrams. TPP participant institutions developed diverse solutions to overcome many barriers, but the use of Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines provided some consistency among the institutions. The TPP also collected some pharmacogenetic implementation outcomes (scientific, educational, financial, and informatics), which may inform healthcare systems seeking to implement their own pharmacogenetic testing programs.

Metabolic activation of R,S-1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur) to 5-fluorouracil by soluble enzymes.

There are two major R,S-1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur) activation pathways to 5-fluorouracil, one that is mediated by microsomal cytochrome P-450 oxidation at C-5' of the tetrahydrofuran moiety and one that is mediated by soluble enzymes. This report demonstrates that the soluble enzyme pathway proceeds via enzymatic cleavage (possibly hydrolytic) of the N-1--C-2' bond to yield 5-fluorouracil and 4-hydroxybutanal, which is immediately further metabolized to gamma-butyrolactone or gamma-hydroxybutyric acid. The soluble activation pathway was present in liver, small intestine, and brain. Because of the limited distribution of cytochrome P-450 in body tissues and because of the lack of redistribution of 5-fluorouracil via the systemic circulation after ftorafur administration, we propose that the soluble enzyme pathway is at least in part responsible for organ toxicity and possibly antitumor effect. Distinction of the microsomal (C-5') and the soluble enzyme (C-2') activation pathways can be exploited in the design of more selective prodrug analogues.

Distinct effects of adenine and guanine starvation on DNA synthesis associated with different pool sizes of nucleotide precursors.

Guanine nucleotide depletion primarily causes a drastic inhibition of DNA synthesis, while adenine nucleotide depletion interferes with other vital functions before inhibiting DNA synthesis (M. B. Cohen and W. Sadée, Cancer Res., 43: 1589-1591, 1983). This study addresses the hypothesis that the presence of a large adenine nucleotide pool with direct access to DNA synthesis prevents immediate cessation of DNA synthesis under conditions of adenine starvation, while the small guanine-DNA precursor pool is readily exhausted under guanine starvation. Adenine, guanine, and deoxyadenosine tracers were incubated with asynchronized or synchronized S-49 cells, and tracer progression into cellular nucleotide pools and nucleic acids was measured. Compartmentation of the dATP pool into a functional DNA precursor pool and a general cellular pool could not be demonstrated with [3H]dAdo tracer experiments with S-phase cells. While guanine tracer was incorporated into DNA without delay (less than 5 min), consistent with a small functional guanine-DNA precursor pool, adenine tracer incorporation into DNA was associated with a substantial delay period (approximately 30 min) indicative of a large functional adenine-DNA precursor pool. These results suggest that the different size of the functional nucleotide precursor pools with rapid access to DNA synthesis accounts for the dramatic difference in the effects of purine antimetabolites that cause either adenine or guanine starvation.

Interaction among the distinct effects of adenine and guanine depletion in mouse lymphoma cells.

Toxic guanine depletion was shown previously to result in a dramatic reduction of DNA synthesis, while toxic adenine depletion failed to affect DNA synthesis (M. B. Cohen and W. Sadée, Cancer Res., 43: 1587-1591, 1983). In this study, relative DNA synthesis rates were measured in mouse lymphoma S49 cells over 24 hr after drug exposure and were compared to cell growth curves. DNA synthesis inhibition by mycophenolic acid (guanine starvation) was achieved at lower drug concentrations than was the inhibition of cell growth. This result further supports the hypothesis (reference above) that guanine starvation specifically affects cells in S phase while it allows cells with full DNA complement to divide. In contrast, L-alanosine (adenine starvation) failed to affect DNA synthesis for at least 24 hr at a concentration that inhibits cell growth by 80%. The dramatically different effects of guanine and adenine starvation on DNA synthesis can thus be used to assess the magnitude of each when blocking early de novo purine biosynthesis by 6-methyl-mercaptopurine ribonucleoside (6- MMPR ). The results suggest that, although 6- MMPR effects primarily resemble those of guanine depletion, adenine starvation measurably contributes to the overall toxicity of 6- MMPR . Drug combination experiments with L-alanosine, mycophenolic acid, and 6- MMPR suggest that the basic mechanisms underlying the toxic effects of guanine and adenine starvation act synergistically.

Hydroxylated metabolies of R,S-1-(tetrahydro-2-furanyl)-5-fluorouracil (Ftorafur) in rats and rabbits.

Two hydroxylated metabolies (M1 and M2) have been isolated from rabbit urine after administration of Ftorafur (FT). The structures of 3'-OH-FT and 4'-OH-FT were assigned to M1 and M2, respectively. A reverse-phase high performance liquid chromatography assay was developed for jeasuring FT, M1, M2, and 5-fluorouracil (FU) plasma levels. M1, M2, and FU were present in rabbit and rat plasma in greatly varying concentrations after FT administration. Pharmacokinetic studies suggest that FU formation proceeds via metabolic intermediate(s) and that the extent of FT activation is variable. A horse liver thymidine phosphorylase ,reparation capable of catalyzing the conversion of beta-ribo-2'-deoxy-5-fluorouracil to FU was inactive against FT and M1. However, 20% of M2 was converted to FU by this enzume, which suggests that the urinary metabolite M2 consisted of a mixture of enantiomers with 20% present in the natural beta-D configuration. The stereochemistry of M1 remains unknown. Hydroxylation of FT to beta-D-4'-OH-FT and subsequent cleavage to FU by thymidine phosphorylase represents one possible activation mechanism of FT to FU. ,owever, lack of correlation between plasma levels of M2 and FU indicates that this mode of metabolic activation may account for only part of the overall activation of FT in vivo.

Modulation of 6-thioguanine activity by guanine in human promyelocytic leukemia HL-60 cells.

The effects of guanine coadministration on the metabolism and biological activity of 6-thioguanine (6-TG) were studied in human promyelocytic leukemia cells (HL-60). Cell growth, cytotoxicity (cloning assay), and cell differentiation were measured, along with nucleotide metabolism. Guanine was efficiently salvaged by HL-60 cells; at 200 microM, guanine suppressed the formation of 6-TG mononucleotides and abolished 6-TG incorporation into nucleic acids. Similarly, guanine antagonized 6-TG cytotoxicity in a dose dependent fashion. Furthermore, guanine (200 microM) fully suppressed the 6-TG (10 microM) induced HL-60 cell differentiation, which suggests that cell differentiation at pharmacological 6-TG concentrations is dependent on the anabolism of the drug to active nucleotides. 6-TG given alone reduced GTP levels and DNA synthesis rates in HL-60 cells, while a major intracellular 6-TG metabolite, 6-thioguanosine 5'-monophosphate, accumulated to high levels (approximately 100 microM). It is suggested that accumulation of 6-thioguanosine 5'-monophosphate and a resultant partial block of the de novo biosynthesis of guanine nucleotides is responsible for 6-TG induced cell differentiation in HL-60 cells.

An oligopeptide transporter is expressed at high levels in the pancreatic carcinoma cell lines AsPc-1 and Capan-2.

Carcinomas of the exocrine pancreas are poorly understood and have a poor prognosis because of their highly malignant nature. Using two human pancreatic cancer cell lines, AsPc-1 and Capan-2, we have investigated avenues that might be useful in targeting the delivery of antineoplastic agents to such cancers. Qualitative RNA PCRs established the presence of the oligopeptide transporter PEPT 1 in these pancreatic cell lines. Northern analysis confirmed the presence of a 3.3-kb transcript. The transporter is normally expressed primarily in small intestinal epithelial cells for nutrient absorption. It is also expressed in a human intestinal cell line, Caco-2. High levels of PEPT 1 protein expression in AsPc-1 and Capan-2, as multiple glycosylated forms (Mr approximately 90,000-120,000), were confirmed by Western immunoblotting, when compared with Caco-2 cell cultures. Absorption of the model dipeptide glycyl-L-sarcosine by AsPc-1 and Capan-2 cells was similar to glycyl-L-sarcosine absorption by Caco-2 cells and a Chinese hamster ovary cell line expressing human PEPT 1 (CHO-PEPT 1). Uptake was pH dependent and inhibited by several di/tripeptides and bestatin, but it remained unaffected by glycine and tetraglycine. Peptide solute transport by AsPc-1 and Capan-2 cells exhibited binding affinities (Kms) similar to those previously reported for PEPT 1, whereas the transport maximal velocity (Vmax) of the AsPc-1 cells was much greater than those of the Capan-2 and Caco-2 cells. Immunomicroscopy demonstrated PEPT 1 protein localized at the plasma membrane and in intracellular vesicular structures, similar to that observed for Caco-2 and CHO-PEPT 1 cells. These data suggest that the pancreatic cancer cells AsPc-1 and Capan-2 express surprisingly high levels of a solute transporter that was previously thought to be restricted in function to the absorption of nutrients from the small intestine.

Regulation of RNA- and DNA-directed actions of 5-fluoropyrimidines in mouse T-lymphoma (S-49) cells.

The mouse T-lymphoma (S-49) cell line is useful for individually studying RNA- and DNA-directed effects of 5-fluoropyrimidines. On the basis of their metabolic activation, biochemical effects on pyrimidine nucleotide metabolism, and biological toxicity, we hve established that incubation of S-49 cells with 5-fluorodeoxyuridine produces only DNA-directed toxicity (thymidylate synthetase inhibition), incubation with 5-fluorouracil (FUra) + thymidine only RNA-directed toxicity, and incubation with FUra alone produces both DNA- and RNA-directed toxicity. The DNA component of 5-fluoropyrimidine toxicity causes immediate growth inhibition of asynchronous S-49 cell cultures, which is self-limited within 12 hr both by the accumulation of intracellular deoxyuridine 5'-monophosphate competing for thymidylate synthetase binding and by the excretion of deoxyuridine into the cell medium which competes with 5-fluorodeoxyuridine uptake. The RNA-directed component causes growth inhibition and cell kill after a delay of 1 doubling time in asynchronous cultures. Studies with cells synchronized by centrifugal elutriation indicate that the RNA-directed FUra effects are expressed only in the G1 phase of the cell cycle and cause rapid cell lysis, while the DNA-directed component is specific to the S phase. Experiments using continuous exposure of synchronized cells to FUra alone demonstrate that the activities of the RNA- and DNA-directed components interact with each other. Specifically, DNA-directed toxicity arrests cells in S phase, preventing them from progressing into G1 where RNA-directed toxicity is expressed, which may account for the augmentation of FUra toxicity by thymidine as reported in other systems.

Biochemical differences among four inosinate dehydrogenase inhibitors, mycophenolic acid, ribavirin, tiazofurin, and selenazofurin, studied in mouse lymphoma cell culture.

The mechanism of the cellular toxicity of four inosinate dehydrogenase (IMP-DH) inhibitors with different antitumor and antiviral pharmacological profiles was investigated in mouse lymphoma (S-49) cell culture. Drug effects on cell growth, nucleotide pools, and DNA and RNA synthesis were measured in the presence and absence of guanine salvage supplies. Both guanine and guanosine were capable of bypassing the IMP-DH block, while they also demonstrated some growth-inhibitory effects when added alone in high concentrations. All four drugs reduced cellular guanosine triphosphate levels and caused secondary changes of the uridine, cytidine, and adenosine triphosphate pools that were similar among the four drugs. However, several drug effects in addition to IMP-DH inhibition were observed except with mycophenolic acid which may represent a pure IMP-DH inhibitor. Both tiazofurin and selenazofurin interfered with the uptake and/or metabolism of uridine and thymidine tracers; however, this effect appeared not to contribute to their cellular toxicity in vitro. Moreover, selenazofurin and tiazofurin impaired the utilization of exogenous guanine salvage supplies for DNA and RNA synthesis, and guanine was particularly ineffective in reversing the toxic effects of tiazofurin on cell growth. This finding is important in view of the available guanine salvage supplies in vivo. Since tiazofurin, selenazofurin, and their known metabolites failed to inhibit hypoxanthine-guanine-phosphoribosyl transferase, guanosine monophosphate kinase, and guanosine diphosphate kinase in cell extracts or permeabilized cells, these drugs may interfere with salvage transport across cellular membranes. The toxic effects of mycophenolic acid and ribavirin were similarly reversed by salvage supplies of up to 200 microM guanine, which suggests that ribavirin primarily acts as an IMP-DH inhibitor under these conditions. This result could explain the rather low antitumor efficacy of both mycophenolic acid and ribavirin in vivo. However, increasing the guanine salvage supply in the medium above 200 microM further reversed the toxic effects of mycophenolic acid to maximum rescue, while it increased the toxicity of ribavirin (300 microM). This finding suggests the presence of a toxic mechanism of ribavirin at higher concentrations that is dependent upon the presence of guanine supplies sufficient to fully overcome the IMP-DH inhibition. This study documents that each antimetabolite displays a unique spectrum of activities with multiple toxic targets.

Neuronal cell differentiation of human neuroblastoma cells by retinoic acid plus herbimycin A.

We investigated the effect of retinoic acid (RA) and herbimycin A (herb-A) on cell growth, cell differentiation, and colony formation of human neuroblastoma cell lines. The neuroblastoma line SK-N-SH expressed both neuroblast and nonneuronal phenotypes, whereas its subclone SH-SY5Y and the Kelly cell line were predominantly neuroblastic. Both herb-A and RA, given alone, moderately reduced cell growth and colony formation of the neuroblastic cell lines. Growth curve analyses with SK-N-SH suggested that herb-A greatly reduced the number of initially growing cells, whereas RA slightly enhanced initial cell growth. Morphological changes were determined with the use of rhodaminephalloidin staining of actin. Retinoic acid caused an increase in the fraction of neuroblast cell in SK-N-SH, and conversely of nonneuronal cells in SH-SY5Y and Kelly cell lines. Both drugs also caused partial differentiation towards a neuronal phenotype, and herb-A induced selective lysis of nonneuronal cells of SK-N-SH. Because of their discrepant effects, RA (10 microM) and herb-A (236 nM) were tested in combination at a concentration that had only moderate effects when given alone. The combination further reduced cell growth and colony formation and dramatically enhanced differentiation towards a neuronal morphology. The Kelly cell line with amplified N-myc genome, which correlates with clinical progression of neuroblastoma, was also sensitive to RA plus herb-A. These results recommend the combination of RA and herb-A for differentiation therapy of neuroblastoma.

Expression of neurotransmitter receptors and myc protooncogenes in subclones of a human neuroblastoma cell line.

Phenotypic variability of the human neuroblastoma cell line SK-N-SH was studied with the use of three subclones that interconvert at a slower rate than the parent cell line, i.e., a neuroblast-type subclone (SH-SY5Y), a nonneuronal, strongly substrate adherent subclone (SH-EP), and an intermediate type subclone (SH-IN). Rhodamine-phalloidin staining of actin fibers revealed differences in the cytoskeleton morphology of the three subclones, while the clathrin subunit proteins (heavy and light chains), components of coated vesicles, were invariant. Dramatic differences were observed for the expression of neurotransmitter systems, i.e., the mu and delta opioid receptor, the muscarinic cholinergic receptor and its effect on phosphatidylinositol turnover, and the uptake1 transporter for catecholamines. While these systems were strongly expressed in the parent line and the neuroblast-like clones SH-SY5Y and SH-IN, they were absent or barely detectable in the nonneuronal EP clone. Furthermore, the protooncogenes N- and c-myc were only expressed in the neuroblast containing lines, consistent with their growth characteristics of fully transformed cells. The strong c-myc expression in the absence of c- or N-myc amplification in SK-N-SH, adds a new form of high protooncogene activity in neuroblastoma cell lines. The remarkable differences of neurotransmitter systems and myc expression among the various phenotypes of human neuroblastoma cells should be considered in the therapy of neuroblastoma.

Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for CYP3A5 Genotype and Tacrolimus Dosing.

Tacrolimus is the mainstay immunosuppressant drug used after solid organ and hematopoietic stem cell transplantation. Individuals who express CYP3A5 (extensive and intermediate metabolizers) generally have decreased dose-adjusted trough concentrations of tacrolimus as compared with those who are CYP3A5 nonexpressers (poor metabolizers), possibly delaying achievement of target blood concentrations. We summarize evidence from the published literature supporting this association and provide dosing recommendations for tacrolimus based on CYP3A5 genotype when known (updates at www.pharmgkb.org).

Effects of spironolactone, canrenone and canrenoate-K on cytochrome P450, and 11beta- and 18-hydroxylation in bovine and human adrenal cortical mitochondria.

Effects of spironolactone, canrenone and canrenoate-K on adrenal cytochrome P450 (P450) and corticosteroid biosynthesis were examined by studying difference spectra, P450 reduction and corticoid hydroxylation in mitochondrial preparations isolated from zona fasciculata and zona glomerulosa of bovine adrenals and from adrenal adenoma and hyperplastic adrenal cortex removed from patients with hyperaldosteronism. All three agents bound to P450 producing type I difference spectra and underwent hydroxylation. They all inhibited 11beta-hydroxylation in bovine adrenal at 30 muM and higher concentrations. Canrenone, the most potent inhibitor, blocked enzyme activity by 60% at a concentration of 60 muM. Spironolactone stimulated P450 reduction. The order of potency of inhibition was found to correlate with the order of affinity of these agents for P450. 11beta-Hydroxylase in human adrenal appeared to be less sensitive to canrenone. All three agents or their hydroxylated metabolites blocked 18-hydroxylation in bovine adrenal at lower concentrations. Canrenoate-K, being the most effective, inhibited 52% at 20 muM. Low concentrations of canrenone (2.5-5.0 muM) were without effect on 11beta-hydroxylase but markedly inhibited 18-hydroxylation (62-76%) in hyperplastic human adrenals. The inhibitors produced mixed type inhibition of 11beta-hydroxylation and competitive type inhibition of 18-hydroxylation. These findings indicate that at low concentrations spironolactone and its major metabolites, canrenone and canrenoate-K, or their hydroxylated metabolites, can directly interfere with the biosynthesis of aldosterone in bovine and certain human adrenal cortical tissue.

Basal phosphorylation of mu opioid receptor is agonist modulated and Ca2+-dependent.

The mu opioid receptor was shown to be phosphorylated at a basal rate in the absence of agonist, measured in permeabilized HEK293 cells transfected with an epitope tagged mu receptor (EE-mu) [Arden, J., Segredo, V., Wang, Z., Lameh, J. and Sadee, W. (1995) J. Neurochem. 65, 1636-1645]. In the present study, basal phosphorylation was found to be Ca2+ dependent; however, several inhibitors of protein kinase C and Ca2+-calmodulin dependent kinases failed to affect basal mu receptor phosphorylation. Thus, the basal mu receptor phosphorylating activity differed from the main kinases involved in receptor regulation. The general kinase inhibitor H7 (100 microM) suppressed basal mu receptor phosphorylation. Pretreatment with the agonist morphine, followed by drug removal, resulted in a sustained increase of basal mu receptor phosphorylation. The gradual agonist dependent modulation of basal mu receptor phosphorylation suggests a novel regulatory mechanism which may play a role in narcotic tolerance and dependence.

Retinoic acid enhances VIP receptor expression and responsiveness in human neuroblastoma cell, SH-SY5Y.

Retinoic acid (RA) induces partial differentiation of neuroblastoma (NB) cells in vitro. In the human NB line, SH-SY5Y (a neuroblastic subclone of SK-N-SH), RA was previously shown to enhance the stimulatory (PGE1) and inhibitory (opioid) regulation of adenylyl cyclase. Since these cells are also sensitive to cAMP stimulation by vasoactive intestinal peptide (VIP), we have tested the effects of RA on VIP receptor expression and function. Pretreatment of SH-SY5Y cells with 10 microM RA over 6 days dramatically increased VIP receptor number from approximately 3,000 to approximately 70,000 sites per cell and enhanced threefold the cAMP accumulation after external VIP addition, while VIP immunoreactive content in the cells increased 2-3-fold. In the light of the recently proposed autocrine function of VIP in this cell lineage, the strong enhancement of the VIP system may contribute to the differentiation effects of RA.

Differential expression of alpha-subunits of G-proteins in human neuroblastoma-derived cell clones.

The distribution of alpha- and beta-subunits of G-proteins was analyzed in membranes of three cell clones which are derived from the human neuroblastoma cell line SK-N-SH. The neuroblast-like clone SH-SY5Y shows a pattern of G-proteins very similar to that of human brain cortex with high levels of Gi alpha and Go alpha but low levels of G40 alpha. The intermediate clone SH-IN contains high levels of Go alpha and Gi alpha and moderate levels of G40 alpha. The non-neuronal clone SH-EP shows high levels of G40 alpha but lacks Go alpha. Differentiation of the neuroblast-like clone SH-SY5Y by retinoic acid or nerve growth factor does not change the amount of Gi alpha or Go alpha in the membrane.