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NPM 1-mutated acute myeloid leukemia (AML) shows unique features. However, the characteristics of "therapy-related" NPM1-mutated AML (t-NPM1 AML) are poorly understood. We compared the genetics, transcriptional profile, and clinical outcomes of t-NPM1 AML, de novo NPM1-mutated AML (dn-NPM1 AML), and therapy-related AML (t-AML) with wild-type NPM1 (t-AML). Normal karyotype was more frequent in t-NPM1 AML (n = 78/96, 88%) and dn-NPM1 (n = 1986/2394, 88%) than in t-AML (n = 103/390, 28%; P < .001). DNMT3A and TET2 were mutated in 43% and 40% of t-NPM1 AML (n = 107), similar to dn-NPM1 (n = 88, 48% and 30%; P > 0.1), but more frequently than t-AML (n = 162; 14% and 10%; P < 0.001). Often mutated in t-AML, TP53 and PPM1D were wild-type in 97% and 96% of t-NPM1 AML, respectively. t-NPM1 and dn-NPM1 AML were transcriptionally similar, (including HOX genes upregulation). At 62 months of median follow-up, the 3-year overall survival (OS) for t-NPM1 AML (n = 96), dn-NPM1 AML (n = 2394), and t-AML (n = 390) were 54%, 60%, and 31%, respectively. In multivariable analysis, OS was similar for the NPM1-mutated groups (hazard ratio [HR] 0.9; 95% confidence interval [CI], 0.65-1.25; P = .45), but better in t-NPM1 AML than in t-AML (HR, 1.86; 95% CI, 1.30-2.68; P < .001). Relapse-free survival was similar between t-NPM1 and dn-NPM1 AML (HR, 1.02; 95% CI, 0.72-1.467; P = .90), but significantly higher in t-NPM1 AML versus t-AML (HR, 1.77; 95% CI, 1.19-2.64; P = .0045). t-NPM1 and dn-NPM1 AML have overlapping features, suggesting that they should be classified as a single disease entity., (© 2023 by The American Society of Hematology.)

Nuclear factor I-C (NFIC) belongs to a family of NFI transcription factors that binds to DNA through CAATT-boxes and are involved in cellular differentiation and stem cell maintenance. Here we show NFIC protein is significantly overexpressed in 69% of acute myeloid leukemia patients. Examination of the functional consequences of NFIC overexpression in HSPCs showed that this protein promoted monocytic differentiation. Single-cell RNA sequencing analysis further demonstrated that NFIC overexpressing monocytes had increased expression of growth and survival genes. In contrast, depletion of NFIC through shRNA decreased cell growth, increased cell cycle arrest and apoptosis in AML cell lines and AML patient blasts. Further, in AML cell lines (THP-1), bulk RNA sequencing of NFIC knockdown led to downregulation of genes involved in cell survival and oncogenic signaling pathways including mixed lineage leukemia-1 (MLL-1). Lastly, we show that NFIC knockdown in an ex vivo mouse MLL::AF9 pre-leukemic stem cell model, decreased their growth and colony formation and increased expression of myeloid differentiation markers Gr1 and Mac1. Collectively, our results suggest that NFIC is an important transcription factor in myeloid differentiation as well as AML cell survival and is a potential therapeutic target in AML., (© 2022. The Author(s).)

RUNX3 is a transcription factor dysregulated in acute myeloid leukemia (AML). However, its role in normal myeloid development and leukemia is poorly understood. Here we investigate RUNX3 expression in both settings and the impact of its dysregulation on myelopoiesis. We found that RUNX3 mRNA expression was stable during hematopoiesis but decreased with granulocytic differentiation. In AML, RUNX3 mRNA was overexpressed in many disease subtypes, but downregulated in AML with core binding factor abnormalities, such as RUNX1::ETO. Overexpression of RUNX3 in human hematopoietic stem and progenitor cells (HSPC) inhibited myeloid differentiation, particularly of the granulocytic lineage. Proliferation and myeloid colony formation were also inhibited. Conversely, RUNX3 knockdown did not impact the myeloid growth and development of human HSPC. Overexpression of RUNX3 in the context of RUNX1::ETO did not rescue the RUNX1::ETO-mediated block in differentiation. RNA-sequencing showed that RUNX3 overexpression downregulates key developmental genes, such as KIT and RUNX1, while upregulating lymphoid genes, such as KLRB1 and TBX21. Overall, these data show that increased RUNX3 expression observed in AML could contribute to the developmental arrest characteristic of this disease, possibly by driving a competing transcriptional program favoring a lymphoid fate., (© 2022. The Author(s).)

RUNX proteins belong to a family of transcription factors essential for cellular proliferation, differentiation, and apoptosis with emerging data implicating RUNX3 in haematopoiesis and haematological malignancies. Here we show that RUNX3 plays an important regulatory role in normal human erythropoiesis. The impact of altering RUNX3 expression on erythropoiesis was determined by transducing human CD34 + cells with RUNX3 overexpression or shRNA knockdown vectors. Analysis of RUNX3 mRNA expression showed that RUNX3 levels decreased during erythropoiesis. Functionally, RUNX3 overexpression had a modest impact on early erythroid growth and development. However, in late-stage erythroid development, RUNX3 promoted growth and inhibited terminal differentiation with RUNX3 overexpressing cells exhibiting lower expression of glycophorin A, greater cell size and less differentiated morphology. These results suggest that suppression of RUNX3 is required for normal erythropoiesis. Overexpression of RUNX3 increased colony formation in liquid culture whilst, corresponding RUNX3 knockdown suppressed colony formation but otherwise had little impact. This study demonstrates that the downregulation of RUNX3 observed in normal human erythropoiesis is important in promoting the terminal stages of erythroid development and may further our understanding of the role of this transcription factor in haematological malignancies., (© 2022. The Author(s).)

Monitoring of NPM1 mutant (NPM1mut) measurable residual disease (MRD) in acute myeloid leukemia (AML) has an established role in patients who are treated with intensive chemotherapy. The European LeukemiaNet has defined molecular persistence at low copy number (MP-LCN) as an MRD transcript level <1% to 2% with a <1-log change between any 2 positive samples collected after the end of treatment (EOT). Because the clinical impact of MP-LCN is unknown, we sought to characterize outcomes in patients with persistent NPM1mut MRD after EOT and identify factors associated with disease progression. Consecutive patients with newly diagnosed NPM1mut AML who received ≥2 cycles of intensive chemotherapy were included if bone marrow was NPM1mut MRD positive at the EOT, and they were not transplanted in first complete remission. One hundred patients were followed for a median of 23.5 months; 42% remained free of progression at 1 year, either spontaneously achieving complete molecular remission (CRMRD-; 30%) or retaining a low-level NPM1mut transcript (12% for ≥12 months and 9% at last follow-up). Forty percent met the criteria for MP-LCN. Preemptive salvage therapy significantly prolonged relapse-free survival. Risk factors associated with disease progression were concurrent FLT3-internal tandem duplication at diagnosis and suboptimal MRD response (NPM1mut reduction <4.4-log) at EOT., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Treatment of relapsed/resistant acute myeloid leukaemia (AML) remains a significant area of unmet patient need, the outlook for most patients remaining extremely poor. A promising approach is to augment the anti-tumour immune response in these patients; most cancers do not activate immune effector cells because they express immunosuppressive ligands. We have previously shown that CD200 (an immunosuppressive ligand) is overexpressed in AML and confers an inferior overall survival compared to CD200low/neg patients. Here we show that a fully human anti-CD200 antibody (TTI-CD200) can block the interaction of CD200 with its receptor and restore AML immune responses in vitro and in vivo., (© 2020 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Inappropriate localization of proteins can interfere with normal cellular function and drive tumor development. To understand how this contributes to the development of acute myeloid leukemia (AML), we compared the nuclear proteome and transcriptome of AML blasts with normal human CD34 + cells. Analysis of the proteome identified networks and processes that significantly affected transcription regulation including misexpression of 11 transcription factors with seven proteins not previously implicated in AML. Transcriptome analysis identified changes in 40 transcription factors but none of these were predictive of changes at the protein level. The highest differentially expressed protein in AML nuclei compared with normal CD34 + nuclei (not previously implicated in AML) was S100A4. In an extended cohort, we found that over-expression of nuclear S100A4 was highly prevalent in AML (83%; 20/24 AML patients). Knock down of S100A4 in AML cell lines strongly impacted their survival whilst normal hemopoietic stem progenitor cells were unaffected. These data are the first analysis of the nuclear proteome in AML and have identified changes in transcription factor expression or regulation of transcription that would not have been seen at the mRNA level. These data also suggest that S100A4 is essential for AML survival and could be a therapeutic target in AML.

Although the prognostic impact of mutations of FLT3 and NPM1 have been extensively studied in younger patients with acute myeloid leukaemia, less is known in older patients whether treated intensively or non-intensively, or in the context of existing prognostic scores. In 1312 patients 16 and 21%, respectively had an FLT3 and NPM1 mutation. An FLT3 mutation did not affect remission rate in intensively or non-intensively treated patients but was associated with an inferior survival. All patients with an NPM1c mutation had a significantly higher remission rate irrespective of treatment approach but survival was not improved, overall, or in any genotype except as in younger patients, in the FLT3 WT NPM1c mutant subgroup. When incorporated into an established multi-parameter prognostic risk score, the molecular information provided additional prognostic definition in 11% of patients.

Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5-d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r(2) correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.

Leukemia-associated chimeric oncoproteins often act as transcriptional repressors, targeting promoters of master genes involved in hematopoiesis. We show that CRABPI (encoding cellular retinoic acid binding protein I) is a target of PLZF, which is fused to RARalpha by the t(11;17)(q23;q21) translocation associated with retinoic acid (RA)-resistant acute promyelocytic leukemia (APL). PLZF represses the CRABPI locus through propagation of chromatin condensation from a remote intronic binding element culminating in silencing of the promoter. Although the canonical, PLZF-RARalpha oncoprotein has no impact on PLZF-mediated repression, the reciprocal translocation product RARalpha-PLZF binds to this remote binding site, recruiting p300, inducing promoter hypomethylation and CRABPI gene up-regulation. In line with these observations, RA-resistant murine PLZF/RARalpha+RARalpha/PLZF APL blasts express much higher levels of CRABPI than standard RA-sensitive PML/RARalpha APL. RARalpha-PLZF confers RA resistance to a retinoid-sensitive acute myeloid leukemia (AML) cell line in a CRABPI-dependent fashion. This study supports an active role for PLZF and RARalpha-PLZF in leukemogenesis, identifies up-regulation of CRABPI as a mechanism contributing to retinoid resistance, and reveals the ability of the reciprocal fusion gene products to mediate distinct epigenetic effects contributing to the leukemic phenotype.

The FUS gene is overexpressed in acute myeloid leukaemia (AML) patients and has roles in transcription and mRNA processing. We used ectopic expression of FUS and FUS antisense sequences to assess the effect of modulation of FUS expression in all-trans retinoic acid (ATRA)-sensitive (NB4) and insensitive (NB4R2) human acute promyelocytic (APL) cell lines which express the t(15:17) translocation. Growth, viability and differentiation patterns were maintained, but the expression of the FUS antisense construct in both the cell lines altered the response to ATRA: the previously ATRA-sensitive NB4 cells exhibited resistance; whilst the previously resistant NB4R2 cells showed a differentiation response to treatment.

The receptor tyrosine kinase FLT3 is a promising molecular therapeutic target in acute myeloid leukemia (AML). Activating mutations of FLT3 are present in approximately one-third of patients, while many nonmutants show evidence of FLT3 activation, which appears to play a significant role in leukemogenesis. We studied the effects of lestaurtinib (CEP701) and PKC412, 2 small molecule inhibitors of FLT3, on 65 diagnostic AML blast samples. Both agents induced concentration-dependent cytotoxicity in most cases, although responses to PKC412 required higher drug concentrations. Cytotoxic responses were highly heterogeneous and were only weakly associated with FLT3 mutation status and FLT3 expression. Importantly, lestaurtinib induced cytotoxicity in a synergistic fashion with cytarabine, particularly in FLT3 mutant samples. Both lestaurtinib and PKC412 caused inhibition of FLT3 phosphorylation in all samples. Translation of FLT3 inhibition into cytotoxicity was influenced by the degree of residual FLT3 phosphorylation remaining and correlated with deactivation of STAT5 and MAP kinase. FLT3 mutant and wild-type cases both varied considerably in their dependence on FLT3 signaling for survival. These findings support the continued clinical assessment of FLT3 inhibitors in combination with cytotoxic chemotherapy: Entry to future clinical trials should include FLT3 wild-type patients and should remain unrestricted by FLT3 expression level.

Since the first demonstration in 1999 that gene expression profiling could distinguish between different variants of acute leukemia, several studies have analyzed patients with acute myeloid leukemia on the basis of cytogenetics, morphologic subgroups, secondary mutations such as FLT3, prognosis, and therapeutic response. This review examines some of these data and attempts to discuss whether these analyses will have clinical applications in diagnosis, prediction of prognosis and response to therapy, disease classification, or individually targeted therapy. It is probable that all these areas will reach the clinical environment eventually, but in the short to medium term, microarrays will be involved only in diagnosis.

The prognostic significance of FLT3 mutations in acute promyelocytic leukemia (APL) is not firmly established and is of particular interest given the opportunities for targeted therapies using FLT3 inhibitors. We studied 203 patients with PML-RARA-positive APL; 43% of the patients had an FLT3 mutation (65 internal tandem duplications [ITDs], 19 D835/I836, 4 ITD+D835/I836). Both mutations were associated with higher white blood cell (WBC) count at presentation; 75% of the patients with WBC counts of 10 x 10(9)/L or greater had mutant FLT3. FLT3/ITDs were correlated with M3v subtype (P < .001), bcr3 PML breakpoint (P < .001), and expression of reciprocal RARA-PML transcripts (P = .01). Microarray analysis revealed differences in expression profiles among patients with FLT3/ITD, D835/I836, and wild-type FLT3. Patients with mutant FLT3 had a higher rate of induction death (19% vs 9%; P = .04, but no significant difference in relapse risk (28% vs 23%; P = .5) or overall survival (59% vs 67%; P = .2) at 5 years. In in vitro differentiation assays using primary APL blasts (n = 6), the FLT3 inhibitor CEP-701 had a greater effect on cell survival/proliferation in FLT3/ITD+ cells, but this inhibition was reduced in the presence of ATRA. Furthermore, in the presence of CEP-701, ATRA-induced differentiation was reduced in FLT3/ITD+ cells. These data carry implications for the use of FLT3 inhibitors as frontline therapy for APL.

Acute myeloid leukaemia (AML) is a difficult to treat disease, especially for those patients who have no eligible haematopoietic stem cell (HSC) donor. One of the most promising treatment options for these patients is immunotherapy. To investigate the expression of known tumour antigens in AML, we analysed microarray data from 124 presentation AML patient samples and investigated the present/absent calls of 82 tumour-specific or -associated antigens. We found 11 antigens which were expressed in AML patient samples but not normal donors. Nine of these were cancer-testis (CT) antigens, previously shown to be expressed in tumour cells and immunologically protected sites and at very low levels, if at all, in normal tissues. Expression was confirmed using real-time PCR. We have identified a number of CT antigens with expression in presentation AML samples but not normal donor samples, which may provide effective targets for future immunotherapy treatments early in disease.

Mutations of the FLT3 gene, a receptor tyrosine kinase, are the most frequent genetic alteration reported in acute myeloid leukaemia, with internal tandem duplications (ITD) or mutations within the activating loop (AL) reported at a frequency of around 24% and 6%, respectively. ITD mutations have associated with a poor prognosis. In this study we have used polymerase chain reaction (PCR), combined with restriction enzyme digestion for the detection of AL mutations, with the DNA products separated on the Agilent 2100 Bioanalyser using a DNA-500 kit. This analysis enabled the rapid identification of mutations in FLT3, approximate sizing of the ITD, an estimate of the proportion of mutant RNA and in some cases, specific heteroduplex patterns associated with triplet deletions. Our data shows that approximately 16% of the patients examined had an ITD mutation and over 13% had a mutation in the AL including triplet deletions involving codons 835/836 and point mutations in codon D839. Based on the sensitivity and speed of the bioanalyser, we suggest that this method is invaluable and provides an improvement to the current use of agarose gels for the analysis of FLT3 PCR products.

The Philadelphia (Ph) chromosome, a characteristic cytogenetic marker of chronic myeloid leukaemia (CML), is caused by a reciprocal translocation juxtaposing the 3' region of the ABL gene onto the 5' region of the BCR gene. Due to conservation of the reading frame, but depending on the site of the breakpoint in the BCR gene, two alternatively spliced variants of the p210BCR-ABL mRNA (known as b2-a2 and b3-a2) are produced. To investigate whether there are any biological differences between these splice variants we have transfected the b3-a2 or b2-a2 cDNA into a murine myeloid cell line, 32D. We have also included the previously prepared 32Dp210 cell line (which expresses the b3-a2 transcript) in all of our comparisons. RT-PCR analysis indicated that transcription levels were comparable between the variants. Morphological examination of the cells expressing either of the BCR-ABL transcripts indicated that these cells were more mature with increased cytoplasm:nuclear ratios compared to the 32D parental and 32Dneo vector control cells. However, the 32Dp210 cells had a very different appearance from the other panel members and flow karyotyping indicated a clonal evolution and cytogenetic instability in these cells alone. At 10(6) and 10(7) cell doses all 32D cells expressing BCR-ABL caused ill health and tissue infiltration in SCID mice with such rapidity that statistical analysis was not informative. However, at the 10(5) and 10(4) dosage levels there were similar survival rates between mice injected with 32Db2-a2 or 32Db3-a2 while mice injected with 32Dp210 had a significantly shorter survival time. The study of this 32D cell line panel indicated that there were no overt differences in the biological properties conferred by the b3-a2 or b2-a2 transcripts to the 32D cells although these transcripts were able to confer in vitro and in vivo biological effects. This panel of BCR-ABL expressing 32D cells provides a useful model for CML disease progression studies.

Retinoic acid has the ability to induce differentiation in some myeloid leukaemia cell lines and has been used to induce remission in acute promyelocytic leukaemia patients. We have analysed changes in gene expression, by differential display, in HL60 cells exposed to all-trans retinoic acid (ATRA) for only 1 h. Only about 0.4% of the genes examined by this technique showed changes in expression level, and all four of the gene fragments identified were downregulated during the short 1 h exposure. Two of the fragments were novel, a third was MYC and the fourth was the FUS proto-oncogene. Northern analysis showed that FUS was downregulated within 1 h only during induced neutrophil differentiation but not at all during induced monocyte differentiation. Unlike the sensitive cell lines, ATRA-resistant cell lines did not show a downregulation of FUS over a 24 h period of exposure to ATRA. Using a semiquantitative PCR analysis, no difference in FUS levels was observed between ATRA-sensitive and -resistant cell lines. A similar analysis was carried out on primary acute myeloid leukaemia (AML), peripheral stem cell harvests (PBSC) and cord blood samples. The PBSC and cord blood samples had FUS levels that were similar or generally less than the cell lines. However, much higher levels were seen in 63% of the AML samples examined. The data presented are consistent with previous reports for a role for FUS in the promotion and maintenance of cellular proliferation.

The myelomonocytic cell line HL60 can be induced by a variety of chemical agents to differentiation to either neutrophils or monocytes. Examination of gene expression, by differential display, in cells induced to monocytes with 1alpha,25-dihydroxyvitamin D(3) or neutrophils with all-trans retinoic acid (ATRA) identified a number of clones with altered patterns of expression over the period of differentiation. One of these clones was the mitochondrial gene NADH dehydrogenase subunit 4 (ND4) which showed a differential pattern of expression between the neutrophil and monocyte lineages. The potential of mitochondrial inhibitors to induce differentiation was investigated by treating the HL60 cells with either the NADH dehydrogenase inhibitor, Rotenone, the complex III inhibitor, Antimycin A, or the highly specific mitochondrial ATP-synthase inhibitor, Oligomycin. Although functional assays of differentiation did not produce any positive results, all the inhibitors resulted in a dramatic increase in CD14 expression at day 1, with CD38 markers not observed until day 3. The increased expression of CD14 was accompanied by a decrease in viability and all CD14 positive cells were also positive for Annexin V, a marker of apoptosis. These results suggest that inhibition of the components of the mitochondrial pathways may lead to the marking of some cells, via CD14, for cell death, whilst allowing commitment to differentiation to occur in the surviving population., (Copyright 1999 Academic Press.)

Neutrophil and monocyte differentiation can be induced in HL60 leukaemia cells by all-trans-retinoic acid (ATRA) and 1alpha,25-dihydroxyvitamin D3 (D3), respectively, whose differentiating effects can be enhanced by exposure to 'anti-inflammatory agents' and steroids. We have provided evidence that this potentiation is via inhibition of the activity of an enzyme of the aldoketoreductase (AKR) family, but had failed to identify expression of known AKRs in HL60 cells. In this study, we have identified a previously unclassified aldoketoreductase family member (termed HAKR e) that is expressed in HL60 cells. HAKR e is dramatically and transiently up-regulated in HL60 cells within 24 h of exposure to ATRA, further supporting the proposition that a member(s) of this family of enzymes play(s) a role in controlling cell growth and/or differentiation.

A recent clinical therapeutic initiative has been the use of chemical agents which induce the leukaemic cells to overcome their block in differentiation. In order to understand this block the cascade of molecular events needs to be characterized. Haemopoietic differentiation is ultimately controlled at the level of gene transcription which is mediated by an array of transcription factors. Many transcription factors contain similar structural protein sequences, and we have used an RT-PCR-based approach to isolate sequences, from transcription factor gene families which share similar domains. Degenerate primers corresponding to the TFIIIA zinc-finger consensus amino acid sequences and to the POU-homeodomain and POU-specific domain were used to amplify genes on the basis that they contained similarities in structural motifs shared within these families of transcription factors. A serum-independent HL60 cell line was induced towards the neutrophil lineage by treatment with all-trans retinoic acid (ATRA) for 24 h. CD38+ cells committed towards this lineage were enriched and a population of these cells treated with dihydroxyvitamin D3 to induce neutrophil maturation. RNA extracted from uninduced, ATRA-induced CD38+ cells, and vitamin D3 treated maturing cell cultures were amplified using the degenerate primers. PCR fragments were cloned, sequenced, clustered into homologous groups, and the group sequences searched on the GenBank database. The Oct 1 transcription factor, and a very close homologue, KIAA0144, was identified using the POU family primers. The zinc-finger primers identified three zinc-finger genes. The pattern of gene expression was suggested from the number of clones in each group at neutrophil commitment and maturation. The differential expression of the genes in the zinc finger and POU families will lead to a better understanding of the cascade of gene expression which occurs following ATRA-induced differentiation.

Preincubation of AtT-20 mouse pituitary tumour cells with the phorbol ester PMA resulted in a concentration-dependent inhibition of CNP-stimulated cyclic GMP production. The phorbol ester analogue 4 alpha phorbol had no inhibitory effect and 24 h preincubations with PMA resulted in a characteristic down-regulation of the response indicating that the inhibitory actions were mediated via the activation of protein kinase C. Forskolin in the presence of the phosphodiesterase inhibitor IBMX stimulated intracellular cyclic AMP concentrations by up to eight fold, but did not alter basal nor CNP-stimulated cyclic GMP production. These results indicate that CNP-stimulated guanylate cyclase activity associated with the GC-B natriuretic peptide receptor expressed in AtT-20 cells is inhibited by protein kinase C.

Receptors for the natriuretic peptide family have been characterized in the adrenocorticotrophic hormone (ACTH)-secreting AtT-20 pituitary tumour cell line. Northern blot analysis detected mRNA transcripts for the guanylate cyclase-linked GC-B receptor subtype. There was no evidence for the expression of either guanylate cyclase-linked GC-A receptor or atrial natriuretic peptide (ANP)-C (clearance) receptor mRNAs. Cyclic GMP production in AtT-20 cells was stimulated up to 200-fold by C-type natriuretic peptide (CNP), which was 10- and 20 times as effective as equivalent concentrations of brain natriuretic peptide and ANP respectively. Cyclic GMP dose-response curves to CNP failed to show any signs of saturation even at concentrations up to 30 microM, indicating a relatively low affinity of CNP for the GC-B receptor. Although CNP induced large stimulations in cyclic GMP production, specific binding of [125I-Tyr0]CNP could not be demonstrated in AtT-20 cells. The absence of specific binding with this radiolabelled analogue is possibly due to a reduced affinity for the GC-B receptor, as CNP analogues with N-terminal modifications such as [Tyr0]CNP and [127I-Tyr0]CNP exhibited reduced abilities to stimulate cyclic GMP production in these cells. Despite elevating cyclic GMP levels, CNP had no effect on basal or corticotrophin-releasing factor-stimulating ACTH release from the cells. These results show that the guanylate cyclase-coupled GC-B receptor is the only natriuretic peptide receptor subtype expressed in AtT-20 cells. Although CNP can markedly stimulate cyclic GMP production in these cells, there is incomplete expression of the normal natriuretic peptide-induced inhibitory pathway of ACTH secretion at some point distal to the production of cyclic GMP.

Eleven green individuals were isolated when 95000 M2 plants of barley (Hordeum vulgare L.), mutagenised with azide in the M1, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F2 progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Whether atrial natriuretic peptide (ANP)-evoked inhibition of corticotrophin-releasing factor (CRF)-stimulated ACTH secretion was also manifest in ACTH secreting AtT-20 pituitary tumour cells was investigated. ANP stimulated increases in cGMP accumulation at concentrations of the peptide above 10(-8) M which indicates the presence of the ANP receptors on these cells. CRF stimulated a concentration-dependent increase in ACTH secretion from AtT-20 cells which was unaffected by ANP, 8-bromo-cGMP, or sodium nitroprusside (SNP). Calcium stimulated a concentration-dependent increase in ACTH secretion from electrically permeabilised cells which was unaffected by co-incubation with cGMP but potentiated by cAMP. These results reveal the presence of ANP receptors on AtT-20 cells but suggest that an incomplete expression of the stimulus-secretion coupling mechanisms for ANP, at some point after cGMP production, prevents the effects of natriuretic peptides upon ACTH secretion being manifest in these cells.