Proenkephalin assists stress-activated apoptosis through transcriptional repression of NF-kappaB- and p53-regulated gene targets.

The respective pro- and antiapoptotic functions of the transcription factors p53 and nuclear factor kappaB (NF-kappaB), and their potential impact on tumorigenesis and response to tumor therapy are well recognized. The capacity of the RelA(p65) subunit of NF-kappaB to specify a pro-apoptotic outcome in response to some stimuli is less well recognized, but needs to be understood if rational manipulation of the NF-kappaB pathway is to be deployed in cancer therapy. In this report, we provide evidence that the growth-responsive nuclear protein, proenkephalin (Penk), is required, in part, for apoptosis induction, in response to activation or overexpression of p53 and RelA(p65). We describe UV-C-inducible physical associations between endogenous Penk and p53 and RelA(p65) in mammalian cell lines. Depletion of Penk by RNA interference (RNAi) substantially preserves viable cell number following exposure to UV-C irradiation or hydrogen peroxide and confers transient protection in cells exposed to the genotoxin etoposide. In virally transformed and human tumor cell lines, overexpression of nuclear Penk with overabundant or activated p53, or RelA(p65) even in the absence of p53, enhances apoptosis to the point of synergy. We have further shown that Penk depletion by RNAi substantially derepresses transcription of a range of antiapoptotic gene targets previously implicated in repression-mediated apoptosis induction by NF-kappaB and p53. Physical association of endogenous Penk with the transcriptional co-repressor histone deacetylase suggests that it may be a component of a transcriptional repression complex that contributes to a pro-apoptotic outcome, following activation of the NF-kappaB and p53 pathways, and could therefore help to facilitate an antitumor response to a broad range of agents.

HIF-1alpha contributes to tumour-selective killing by the sigma receptor antagonist rimcazole.

We have previously reported tumour-selective killing by the sigma (sigma) receptor ligand rimcazole. We now report that rimcazole elevates hypoxia inducible factor-1alpha (HIF-1alpha) protein levels under normoxic conditions in colorectal (HCT-116) and mammary carcinoma (MDA MB 231) cells but fails to induce HIF-1alpha in normal fibroblasts or mammary epithelial cells. Combining the sigma-1 agonist (+)-pentazocine with rimcazole substantially reduces the accumulation of HIF-1alpha, confirming that the effect is mediated at least partly by antagonism of sigma-1 sites. HIF-1alpha knockdown by RNA interference attenuates rimcazole-induced cell death in both cell types. Thus, the induction of HIF-1alpha by rimcazole contributes to tumour cell killing. In a comparison of HCT-116p53+/+ and HCT-116p53-/- cells, HIF-1alpha levels are consistently higher after rimcazole treatment in HCT-116p53+/+ cells. Furthermore, although rimcazole kills HCT-116p53-/- cells, it has a more potent apoptosis-inducing effect in HCT-116p53+/+ cells. This suggests that the presence of functional p53 protein may enhance death induction by rimcazole in part through greater induction of HIF-1alpha. p53 is not required, however, for the rimcazole-induced engagement of HIF-1alpha in proapoptotic mode as HIF-1alpha knockdown attenuates rimcazole-induced death to comparable extents in p53 mutant and wild-type cell systems. Knowledge of HIF-1alpha involvement may assist the re-profiling of rimcazole and other sigma ligands as cancer therapeutics.

Endogenous opioids and oligodendroglial function: possible autocrine/paracrine effects on cell survival and development.

Previous work has shown that oligodendrocytes (OLs) express both micro- and kappa-opioid receptors. In developing OLs, micro receptor activation increases OL proliferation, while the kappa-antagonist nor-binaltorphimine (NorBNI) affects OL differentiation. Because exogenous opioids were not present in our defined culture medium, we hypothesized that NorBNI blocked endogenous opioids produced by the OLs themselves. To test this, intact and partially processed proenkephalin and prodynorphin-derived peptides were assessed in OLs using immunocytochemistry or Western blot analysis, or both. Immature OLs possessed large amounts of intact and partially processed proenkephalin precursors, as well as posttranslational products of prodynorphin including dynorphin A (1-17). With maturation, however, intact or partially processed proenkephalin was expressed by only about 50% of OLs, while dynorphin A (1-17) was undetectable. To assess the function of OL-derived opioids, the effect of kappa-agonists/antagonists on OL differentiation and death was explored. kappa-Agonists alone had no effect. In contrast, NorBNI significantly increased OL death. Additive OL losses were evident when NorBNI was paired with toxic levels of glutamate, suggesting that kappa-receptor blockade alone is sufficient to induce OL death. Thus, the results indicate that OLs express proenkephalin and prodynorphin peptides in a developmentally regulated manner, and further suggest that opioids produced by OLs modulate OL maturation and survival through local (i.e., autocrine and/or paracrine) mechanisms.

The trigger to cell death determines the efficiency with which dying cells are cleared by neighbours.

Phagocytosis of apoptotic cells is required to prevent tissue injury. Professional phagocytes, such as monocyte-derived macrophages, are highly efficient scavengers of apoptotic cells but their presence cannot always be relied on; in that case, removal of effete cells is accomplished by helpful neighbours. This study describes differences in the efficiency with which apoptotic cells of the same type, but dying in response to different triggers, are engulfed; this varies from engulfment that is so proficient few or no unengulfed apoptotic cells are found, to engulfment that is so delayed apoptotic cells have become secondarily necrotic at the point of engulfment. In all cases the efficiency of engulfment is determined at least in part by the dying cells themselves. p53- and Bax-transfected kidney epithelial (293) cells (transiently transfected using a non-toxic method) were engulfed so proficiently by homotypic neighbours that cells did not show evidence of engagement of the apoptotic programme (chromatin condensation and TUNEL positivity) until engulfment had taken place. Engulfment nonetheless required activation of at least initiator caspases. 293 cells induced to apoptose by other means (etoposide and staurosporine treatment) were not so efficiently ingested: unengulfed apoptotic cells were consistently revealed at all doses and time points, even when treated cells were mixed with healthy, non-treated 293 cells. These data make it extremely unlikely that the fraction of viable, unaffected neighbours determines the efficiency with which engulfment proceeds. Furthermore, 293 cells treated with etoposide or staurosporine were differentially appealing both to homotypic neighbours and to cells in the professional phagocyte lineage (THP-1 cells). If different apoptotic stimuli programme cells to be recognised with different efficiencies, pathways to apoptosis may be injury limiting to greater or lesser degrees.

Proenkephalin is a nuclear protein responsive to growth arrest and differentiation signals.

Neuropeptide precursors are traditionally viewed as molecules destined to be cleaved into bioactive peptides, which are then released from the cell to act on target cell surface receptors. In this report we demonstrate nuclear localization of the enkephalin precursor, proenkephalin, in rodent and human embryonic fibroblasts (Swiss 3T3 and MRC-5 cells) and in rodent myoblasts (C2C12 cells). Nuclear proenkephalin, detected by immunofluorescence with a panel of antiproenkephalin monoclonal antibodies, is distributed predominantly in three patterns. Selective abolition of these patterns with salt, nuclease, or methanol is associated with liberation of immunoprecipitable proenkephalin into the extraction supernatant. Proenkephalin antigenic domains, mapped using phage display libraries and synthetic peptides, are differentially revealed in the three distribution patterns. Selective epitope revelation may reflect different conformational forms of proenkephalin or its existence in complexes with other nuclear proteins, forms which therefore have different biochemical associations with the nuclear substructure. In fibroblast cell populations in transition to growth arrest, nuclear proenkephalin responds promptly to mitogen withdrawal and cell-cell contact by transient, virtually synchronous unmasking of multiple antigenic domains in a fine punctate distribution. A similar phenomenon is observed in myoblasts undergoing differentiation. The acknowledgment of growth arrest and differentiation signals by nuclear proenkephalin suggests its integration with transduction pathways mediating these signals. To begin to address the mechanism of nuclear targeting, we have transfected mutated and nonmutated proenkephalin into COS (African green monkey kidney) cells. Nonmutated proenkephalin is localized exclusively in the cytoplasm; however, proenkephalin mutated at the first ATG codon, or devoid of its signal peptide sequence, is targeted to the nucleus as well as to the cytoplasm. From this we speculate that nuclear proenkephalin arises from a primary translation product that lacks a signal peptide sequence because of initiation at a different site.

Comprehensive epitope analysis of monoclonal anti-proenkephalin antibodies using phage display libraries and synthetic peptides: revelation of antibody fine specificities caused by somatic mutations in the variable region genes.

Filamentous phage libraries, displaying 6, 12 or 20 amino acid residue peptides at the N terminus of coat protein pIII were used to define and localize the epitopes of 15 monoclonal antibodies raised against human proenkephalin, a neuropeptide precursor. Eight monoclonal antibodies (PE14 to PE19, PE23 and PE25), which inhibit each other's binding to proenkephalin, recognized phage clones selected by PE14, PE15, PE19, PE23 and PE25. With the peptide sequences DLL(X)(X)LL (12mer library) and DLL(X)(X)L (6mer library) shared by most of the phage clones it was possible to define the putative antibody epitope 155DLLKELL161 on human proenkephalin. For five antibodies (PE13, PE20 to PE22 and PE24) belonging to another inhibition group, a common consensus motif G(X)D(X)E(X)(X)V(X)(X)R could be defined with help of a 20mer library. The corresponding minimum epitope sequence has been found to be 175GSDNEEEVSKR185. Antibody PE1, raised in a separate fusion, was able to select phage clones from a 12mer and 20mer library, revealing that the sequence 187GGFMRG192 is probably the antibody epitope. The assumed localization of the epitopes was confirmed by screening a set of overlapping synthetic peptides, covering the region of human proenkephalin thought to contain all antibody binding sites. It was found that antibodies, although recognizing the same epitope, gave different binding patterns with the selected phage clones. By analysing the VH chain sequences of these antibodies it could be shown that a varying number of somatic mutations is likely to be the reason for the observed differences in antibody fine specificity.

Ontogeny of proenkephalin mRNA and enkephalin peptide expression in the cerebellar cortex of the rat: spatial and temporal patterns of expression follow maturational gradients in the external granular layer and in Purkinje cells.

Proenkephalin mRNA and peptide products were examined in developing cells of the postnatal rat cerebellar cortex using in situ hybridization and immunocytochemistry. On day 7, proenkephalin mRNA was first detected as discrete cellular labeling in Golgi cells and as a diffuse hybridization signal over the Purkinje cell layer. On day 14, proenkephalin mRNA and peptide products primarily appeared in distinct subpopulations of Purkinje cells present in the posterior and lateral cerebellum. Similarly, in the external granular layer (EGL), enkephalin immunoreactivity was present only in the posterior and lateral portions of the cerebellum on day 14. However, proenkephalin mRNA was not detected in enkephalin-immunoreactive EGL cells. On day 21, the subset of Purkinje cells that expressed proenkephalin mRNA and peptides were distributed more uniformly throughout the cerebellum. On day 28, a few enkephalin-immunoreactive Purkinje cells were uniformly present throughout the cerebellum, but proenkephalin mRNA was not detected in most of these cells. The spatial gradients in proenkephalin mRNA expression evident in the Purkinje cells of younger rats were no longer present in 28-day-old rats. These findings are important, because endogenous opioids such as enkephalin have been previously shown to inhibit the growth of Purkinje cell dendrites and dendritic spines, and inhibit the rate of mitosis in EGL neuroblasts. Cells do not develop at uniform rates within the cerebellum. There are regional differences in the timing of the formation of the EGL, and in the morphogenesis of Purkinje cells. In conjunction with previous work, the present findings suggest that during development, the pattern of enkephalin immunoreactivity in Purkinje and EGL cells closely follows the spatial and temporal gradients of maturation in both these cell types. The emergence and disappearance of enkephalin immunoreactivity in Purkinje and EGL cells is spatially and temporally related, and coincides with proenkephalin mRNA expression in Purkinje cells. Thus, the transient and coordinated appearance of enkephalin in cerebellar Purkinje and EGL cells may contribute to regional differences in the rate of cerebellar maturation, and may help synchronize the developmental interactions between these two cell types.

polo encodes a protein kinase homolog required for mitosis in Drosophila.

We show that mutation in polo leads to a variety of abnormal mitoses in Drosophila larval neuroblasts. These include otherwise normal looking mitotic spindles upon which chromosomes appear overcondensed; normal bipolar spindles with polyploid complements of chromosomes; bipolar spindles in which one pole can be unusually broad; and monopolar spindles. We have cloned the polo gene from a mutant allele carrying a P-element transposon and sequenced cDNAs corresponding to transcripts of the wild-type locus. The sequence shows that polo encodes a 577-amino-acid protein with an amino-terminal domain homologous to a serine-threonine protein kinase. polo transcripts are abundant in tissues and developmental stages in which there is extensive mitotic activity. The transcripts show no obvious spatial pattern of distribution in relation to the mitotic domains of cellularized embryos but are specifically concentrated in dividing cells in larval discs and brains. In the cell cycles of both syncytial and cellularized embryos, the polo kinase undergoes cell cycle-dependent changes in its distribution: It is predominantly cytoplasmic during interphase; it becomes associated with condensed chromosomes toward the end of prophase; and it remains associated with chromosomes until telophase, whereupon it becomes cytoplasmic.

Down-regulation of GAP-43 During Oligodendrocyte Development and Lack of Expression by Astrocytes In Vivo: Implications for Macroglial Differentiation.

The discovery of molecular markers which are selectively expressed during the development of specific classes of rat central nervous system macroglia has greatly advanced our understanding of how these cells are related. In particular, it has been shown in tissue culture that oligodendrocytes and some astrocytes (type-2) may be derived from a common progenitor cell (O-2A progenitor). However, the existence of type-2 astrocytes in vivo has yet to be unequivocally established. Recently, it has been reported that the neural-specific growth-associated protein-43 (GAP-43, otherwise known as B-50, F1, pp46 and neuromodulin) may be expressed by cells of the O-2A lineage in vitro. We set out to examine the cellular specificity of GAP-43 in O-2A progenitors and their descendants in vitro and in vivo. Using a polyclonal antiserum against a GAP-43 fusion protein we have shown the presence of immunoreactive GAP-43 in the membranes of bipotential O-2A glial progenitor cells and type-2 astrocytes by Western blotting and immunocytochemistry of cells in culture. In contrast to previous studies, double labelling with mature oligodendrocyte markers showed that GAP-43 is down-regulated during oligodendrocyte differentiation in vitro. Immunohistochemical staining of sections of developing rat brain demonstrated the same developmental regulation of GAP-43, suggesting that oligodendrocytes only express GAP-43 at immature stages. In addition, normal and reactive astrocytes in tissue sections were not labelled with GAP-43.

A neuropeptide precursor in cerebellum: proenkephalin exists in subpopulations of both neurons and astrocytes.

The adult rat cerebellum has minimal enkephalin immunoreactivity and is devoid of opiate-binding activity. Using novel monoclonal antibodies to the mammalian enkephalin precursor, we describe the immunofluorescent detection of proenkephalin, in the absence of mature enkephalin peptides, in subpopulations of rat cerebellar neurons and astrocytes. In cryostat sections, neurons that express proenkephalin include Golgi cells, macroneurons within deep cerebellar nuclei and a subpopulation of Purkinje cells. Proenkephalin messenger RNA and protein are present in subpopulations of both grey and white matter astrocytes, but not Bergmann glia. In dissociated glial culture, proenkephalin is expressed in process-bearing astrocytes, apparently in association with a subset of intermediate filaments. Proenkephalin within astrocytes is not seen until the second postnatal week and increases through to adulthood. Neuropeptide gene expression adds to the growing range of neuronal-type properties glial cells can display.

Expression of biologically active human pre-procorticotropin releasing hormone in E. coli: characterization and purification.

1. Human pre-procorticotropin releasing hormone (CRH) was expressed in E. coli strain TG2 as a fusion protein with beta-galactosidase. 2. A 140 kDa band which corresponded to beta-galactosidase pre-proCRH fusion protein was identified in lysates of TG2 cells harbouring the recombinant plasmid pre-proCRH (10-196) [ph PPC (10-196)] after sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie Blue staining. The identity of the fusion protein was confirmed by Western blotting and a two-site immunoradiometric assay. 3. Purification of the fusion protein from isolated, washed and solubilized inclusion bodies was achieved by ion-exchange chromatography in the presence of 8 M urea. 4. When comparing the adrenocorticotropin-releasing activity on a molar basis, the potency of the chimeric CRH precursor was 4% of that of synthetic r/h CRH (1-41).

Identification of high molecular weight peptides in bovine adrenomedullary chromaffin granules using monoclonal antibodies to a preproenkephalin A fusion peptide.

Two mouse monoclonal antibodies (PE-1 and PE-2) raised to a beta-galactosidase-preproenkephalin A(69-207) fusion peptide recognize pro-enkephalin A (pro-enk-A) peptides of 33-5 kDa isolated from bovine adrenal chromaffin granules. The preliminary characterization of the high molecular weight adrenomedullary pro-enk-A peptides recognized by PE-1 and PE-2 is described. The high molecular weight peptides were resolved after Sephadex G-50 chromatography and high-performance liquid chromatography (HPLC) into three components (peaks I, II and III). Immunoblot analysis showed each HPLC peak to be heterogeneous. Peak I contained PE-1-and PE-2-immunoreactive peptides of 33, 29, 24 and 22 kDa; peak II contained a peptide of 22 kDa recognized by PE-2, and peptides of 24 and 22 kDa recognized by PE-1; peak III contained a PE-2-immunoreactive peptide of 15 kDa and PE-1-immunoreactive peptide of 18 kDa. Using polyclonal antibodies to peptide F and methionineenkephalin-Arg6-Gly7-Leu8 (MetEnk-RGL), the 22 kDa band cross-reacted with both MetEnk-RGL and peptide F antibodies, whilst the 24 kDa band was shown to possess predominantly MetEnk-RGL immunoreactivity. The 15 kDa (PE-2-immunoreactive) band was recognized by the peptide F but not the MetEnk-RGL antibody, whereas the polyclonal antibodies did not recognize the 18 kDa (PE-1-immunoreactive) band. We propose that the immunological and size characteristics of some of these peptides (29, 24/22, 15 kDa) suggest their similarity to the peptides of predicted molecular mass 23.3, 18.2 and 12.6 kDa previously found in bovine adrenal medulla. The results also indicate the existence of high molecular weight pro-enk-A peptides shortened at the N-terminus. The use of an immunoradiometric assay designed to measure the proenk-A-derived 18.2 kDa peptide using PE-2 and an affinity purified and radioiodinated MetEnk-RGL IgG has supported these findings.

Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms.

Monoclonal antibodies have been generated to a chimeric peptide comprised of Escherichia coli beta-galactosidase fused to the amino acid sequence 69-207 of human preproenkephalin A. Two monoclonal antibodies, PE-1 and PE-2, were identified by their ability to recognize the same segment of proenkephalin A fused to the cII gene product of the E. coli bacteriophage lambda. The binding domains of PE-1 and PE-2 have been broadly located, with respect to the primary translation product, within the amino acid sequences 152-207 and 84-131, respectively. Immunoblot analysis of total bovine adrenomedullary chromaffin granule lysate reveals PE-1 and PE-2 immunoreactive forms of observed molecular mass 35, 33, 29, 24, 22, and 15 kDa, and an 18-kDa PE-1 immunoreactive form. Separation of granule membranes from their contents reveals differential membrane association of these high molecular weight polypeptides. There is preliminary evidence that PE-1 may be detecting a subset of polypeptides where shortening from the NH2 terminus has occurred. We postulate that the 35-kDa form represents the intact bovine enkephalin precursor of predicted molecular mass 27.3 kDa. This experimental approach should be generally applicable to the generation of antibodies which will recognize intact peptide precursors together with their post-translational cleavage products.

The effect of vasopressin infusion on glucose metabolism in man.

Studies on intact animals and isolated rat hepatocytes have shown that arginine vasopression (AVP) stimulates glycogen phosphorylase to break down glycogen and raise plasma glucose concentrations. Since no similar work has been performed on healthy human adults, the effect of moderate (25 pmol/min) and high (75 pmol/min) dose AVP infusion on plasma glucose, intermediary metabolites, glucose kinetics, and circulating glucagon and insulin concentrations was investigated. After AVP infusion, plasma glucose rose from 4.9 +/- 0.1 to a peak of 5.7 +/- 0.2 mmol/l (P less than 0.001), but no changes in blood lactate, pyruvate, alanine, glycerol or 3-hydroxybutyrate concentrations were observed. The glucose rise was accounted for entirely by an increase in the rate of appearance of glucose from 11.19 +/- 0.43 to 13.38 +/- 0.63 mu mol/kg/min (P less than 0.001). Infusion of AVP also increased plasma glucagon concentrations from 38 +/- 8 to 79 +/- 20 pg/l (P less than 0.01). The hyperglycaemic effect of AVP may be mediated solely by stimulation of glucagon release, but we cannot exclude direct stimulation of glycogen phosphorylase activity.

Variation in osmoregulation of arginine vasopressin during the human menstrual cycle.

Osmoregulation of vasopressin secretion was studied in eight healthy women in the follicular and luteal phases of the menstrual cycle. Basal plasma osmolality in the luteal phase was significantly lower than in the follicular period (282.4 +/- 0.6, 285.6 +/- 1.1mmol/kg, respectively, P less than 0.05). Plasma AVP (pAVP) and plasma osmolality (pOsm) were measured during the infusion of 850 mmol/l saline in both phases of the cycle, and linear regression analyses of these data gave the following regression equations (i) follicular, pAVP = 0.43 (pOsm - 284), r = + 0.93, (ii) luteal, pAVP = 0.31 (pOsm - 279), r = + 0.95. Both the slope and abscissal intercept were significantly different (P less than 0.01). Osmotic threshold for thirst sensation in the luteal phase was lower than the follicular (293 +/- 2,297 +/- 1 mmol/kg, P less than 0.005). We conclude that, in the luteal phase, the threshold for AVP release and the gain or sensitivity of the osmostat are reduced together with lowering of the thirst threshold, which account for the lower basal luteal plasma osmolality.

Idiopathic hypergammaglobulinaemia associated with nephrogenic diabetes insipidus and distal renal tubular acidosis.

Renal tubular dysfunction may be recognized in patients suffering from urinary light chain disease or non-myelomatous hypergammaglobulinaemia. We report a patient who has the combination of distal renal tubular acidosis and nephrogenic diabetes insipidus in association with hypergammaglobulinaemia due solely to increased IgG. We postulate that the abnormalities of distal nephron function resulted from cell-mediated immune damage.

The effect of bromocriptine in the polycystic ovary syndrome.

The effect of chronic administration of bromocriptine to 20 patients with the polycystic ovary syndrome (PCOS) was studied. All patients had normal serum prolactin levels. Bromocriptine 7.5 mg daily was administered for up to 1 year and the biochemical and clinical responses assessed at 3-monthly intervals. Over a period of 3 to 12 months significant reductions in serum LH levels, LH/FSH ratios and in plasma testosterone were observed. In 14 patients, blunting of the previously enhanced LH response to LHRH was observed. Clinical improvement also occurred, most notably in menstrual function. In 12 out of 20 patients a monthly cycle was established and in a further three there was an increase in the frequency of menstruation towards normal. Eleven out of 20 patients ultimately noted a subjective improvement in hirsutism. We conclude that bromocriptine, given to patients with polycystic ovarian disease, inhibits LH hypersecretion, leading to restoration of cyclic ovarian function and reduced androgen synthesis.

The regulation of vasopressin secretion in a patient with oat cell carcinoma of the bronchus.

We report a patient who had an oat cell bronchogenic carcinoma in association with the syndrome of inappropriate antidiuresis. There was an unusually long interval between the onset of hyponatraemia and clinically evident malignant disease. Dynamic testing of vasopressin secretion showed preservation of baroregulated, but not osmoregulated, vasopressin release. Immunoreactive vasopressin was detected in pleural fluid, which co-eluted with synthetic vasopressin on gel chromatography.