RESUMEN
The serine/threonine kinase, PIM1, is involved in promoting cell survival in part by phosphorylation and inhibition of proapoptotic proteins. Apoptosis signaling kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, is involved in the so-called stress-activated pathways that contribute to apoptotic cell death. Here we show that PIM1 phosphorylates ASK1 specifically on serine residue 83 (Ser83) both in vitro and in vivo and that PIM1 binds to ASK1 in cells by co-immunoprecipitation. Using H1299 cells, our results further demonstrate that PIM1 phosphorylation of ASK1 decreases its kinase activity induced by oxidative stress. PIM1 phosphorylation of ASK1 on Ser83 inhibited ASK1-mediated c-Jun N-terminal kinase phosphorylation as well as p38 kinase phosphorylation. Under H(2)O(2)-induced stress conditions that normally lead to apoptosis, these phosphorylation events were associated with inhibition of caspase-3 activation and resulted in reduced cell death. Moreover, knockdown of PIM1 in H1299 cells decreased phosphorylation of endogenous Ser83 of ASK1 and was associated with a decrease in cell viability after H(2)O(2) treatment. Taken together, these data reveal a novel mechanism by which PIM1 promotes cell survival that involves negative regulation of the stress-activated kinase, ASK1.
Asunto(s)
Apoptosis/efectos de los fármacos , MAP Quinasa Quinasa Quinasa 5/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/fisiología , Línea Celular Tumoral , Humanos , Estrés Oxidativo/fisiología , Fosforilación , Unión Proteica , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Pim kinases are found to be highly expressed in leukemia, lymphoma, prostate and pancreatic cancer. Bitransgenic mice overexpressing either Pim-1 or Pim-2 and c-Myc succumb to pre-B-cell lymphoma at a strikingly accelerated speed. Despite that Pim-1/Pim-2 has long been recognized as a strong synergistic partner with c-Myc in tumorigenesis, the mechanism underlying the synergism is still not well understood. Overexpression of Pim-1/Pim-2 kinase dramatically stabilizes c-Myc in vivo, and the stabilization is partially mediated by phosphorylation of c-Myc by Pim kinase on a novel site, Ser329. We provide evidence that Pim-2 is more efficient in directly phosphorylating c-Myc Ser329 to stabilize c-Myc. In contrast, we find that Pim-1 is more effective in mediating a decrease in c-Myc Thr58 phosphorylation and an increase in c-Myc Ser62 phosphorylation than in phosphorylating Ser329. In either case, through stabilizing c-Myc, Pim-1/Pim-2 kinases enhance the transcriptional activity of c-Myc. Also knocking down either Pim-1 or Pim-2 dramatically decreases the endogenous levels of c-Myc and thus, its transcriptional activity. Finally, coexpression of the Pim kinases and c-Myc enhances the transforming activity of c-Myc as does the phosphomimic mutant of c-Myc on Ser329. We conclude that these findings appear to explain at least in part the mechanism underlying the synergism between the Pim kinases and c-Myc in tumorigenesis.
Asunto(s)
Genes myc , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Animales , Hidrólisis , Ratones , Ratones Transgénicos , FosforilaciónRESUMEN
The levels of Pim-1, a serine/threonine kinase, increase during phorbol myristate acetate (PMA)-induced myeloid cell differentiation. The tyrosine phosphatase PTP-U2S is also associated with PMA-induced differentiation of myeloid cells and has been shown to enhance differentiation and the onset of apoptosis. PTP-U2S contains a Pim-1 phosphorylation consensus sequence, KKRKLTN, which is efficiently phosphorylated by Pim-1. Immunoprecipitated PTP-U2S from U937 cells was phosphorylated by recombinant Pim-1, resulting in a decrease in its phosphatase activity. During PMA-induced differentiation, U937 cells transfected with the dominant negative Pim-1 underwent rapid differentiation and accelerated apoptosis. The opposite effect was observed for wild-type Pim-1. Our results, therefore, provide compelling evidence that Pim-1 functions to negatively regulate PMA-induced differentiation in part through the phosphorylation of PTP-U2S. Together these data suggest that Pim-1 phosphorylates PTP-U2S in vivo to decrease the phosphatase activity that may be necessary to prevent the premature onset of apoptosis following differentiation.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Apoptosis , Secuencia de Bases , Sitios de Unión/genética , Diferenciación Celular , Cartilla de ADN/genética , Humanos , Cinética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-pim-1 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/metabolismo , Células U937RESUMEN
The cDNA clone of bovine pim-1 has been isolated from phorbol-12-myristate-13-acetate (PMA) and concanavalin A (ConA)-activated peripheral blood lymphocytes (PBLs). The full-length cDNA contains a 411bp 5' untranslated region (5'-UTR), followed by a 939bp coding region and a 3' untranslated region (3'-UTR) that contains 1403bp. Comparison of the bovine pim-1 coding sequence with the human, rat, mouse, frog and zebrafish counterparts reveals 94, 90, 89, 67 and 40% homology at the nucleotide level, respectively. The predicted amino acid sequence of bovine Pim-1 shares 98.7, 97.1, 93.3, 68.8, and 52.4% similarity with the sequences of human, rat, mouse, frog, and zebrafish, respectively. The 5'-UTR of bovine pim-1 shares high sequence similarity to the human and mouse counterparts and is G/C-rich (75%) which may promote a high degree of secondary structure. The 3'-UTR of bovine pim-1 contains two potential polyadenylation sites and an A/T-rich motif which has been shown to decrease the stability of polyA mRNA molecules. Southern blot results indicate that a single copy of the gene exists in the bovine genome. Northern blot results show that PMA stimulation of PBLs increases the expression of the pim-1 mRNA. In addition, examination of Pim-1 protein expression in PBLs stimulated with a variety of mitogens including ConA, PMA, anti-CD3 and purified protein derivative (PPD) from Mycobacterium tuberculosis, reveals two different types of expression patterns during the course of a 24h period of stimulation. ConA and PPD gave a biphasic pattern of expression while PMA and anti-CD3 gave single transient pattern of expression suggesting that expression is controlled by more than one signaling pathway.
Asunto(s)
Bovinos/genética , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/genética , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting/veterinaria , Southern Blotting/veterinaria , Western Blotting/veterinaria , Ciclo Celular/fisiología , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Humanos , Mitógenos/farmacología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas c-pim-1 , ARN/química , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Células U937RESUMEN
Pim-1 belongs to a family of serine/threonine protein kinases that are highly conserved through evolution in multicellular organisms. Originally identified from moloney murine leukemia virus (MuLV)-induced T-cell lymphomas in mice, Pim-1 kinase is involved in the control of cell growth, differentiation and apoptosis. Expression of Pim-1 kinase can be stimulated by a variety of growth factors and regulated at four different levels: transcriptional, post-transcriptional, translational and post-translational. Several signal transduction pathways may be associated with the regulation of Pim-1's expression; accumulating data support that the expression of Pim-1 protein is mediated through activation of JAK/STATs. Recent studies of Pim family kinases indicate that Pim-1 kinase plays important roles outside of the hematopoietic system as well.
Asunto(s)
Apoptosis/fisiología , División Celular/fisiología , Transformación Celular Neoplásica , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Animales , Diferenciación Celular/fisiología , Regulación Enzimológica de la Expresión Génica , Humanos , Linfoma/etiología , Ratones , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-pim-1 , Transducción de SeñalRESUMEN
Human granulocyte-macrophage colony-stimulating factor (GM-CSF), a hemopoietic growth factor, was produced and secreted from tobacco cell suspensions. The GM-CSF cDNA was carried by a binary vector under the control of the CaMV 35S promoter and the T7 terminator. In addition, a 5'-nontranslated region from the tobacco etch virus (TEV leader sequence) was fused to the N-terminal end of the GM-CSF transgene. For ease of purification, a 6-His tag was added to the 3' end of the GM-CSF cDNA. Addition of the TEV leader sequence increased protein production more than twofold compared to non-TEV controls. Initial batch cultivation studies indicated a maximum of 250 microg/L extracellular and 150 microg/L intracellular GM-CSF. Western blot analysis detected multiple peptides with masses from 14 to 30 kDa in the extracellular medium. The plant-produced GM-CSF was biologically active and could be bound to a nickel affinity matrix, indicating that both the receptor-binding region and the 6-His tag were functional. The batch production of GM-CSF was compared with the production of other recombinant proteins secreted by transformed tobacco cells. The recovery of secreted GM-CSF was increased by the addition of stabilizing proteins and by increasing salt in the growth medium to physiological levels.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/aislamiento & purificación , Northern Blotting , Western Blotting , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Histidina/genética , Humanos , Plantas Tóxicas , Plásmidos , Potyvirus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Rhizobium/genética , Nicotiana/citología , Nicotiana/genética , Transformación GenéticaRESUMEN
The lactogen-dependent rat Nb2 lymphoma is a useful model to investigate PRL signaling pathways that lead to regulation of gene transcription. A primary mechanism coupled to PRL receptor (PRLR) activation in Nb2 cells involves phosphorylation by Jak-family tyrosine kinases of one or more signal transducers and activators of transcription (Stat) factors which subsequently bind to gamma-interferon activation sequences (GAS) within promoter regions of target genes. However, it is presently unclear whether this mechanism is operative as a means for regulating PRL-induced gene expression to the exclusion of other signaling pathways. Previously, we reported that PRL directly stimulated rapid expression of the protooncogene, pim-1, at the mRNA and protein levels in lactogen-dependent Nb2-11 cells. In the present study, experiments were conducted to evaluate signaling mechanisms by which PRL regulates transcription of pim-1. Toward this end, a 1,268-bp segment upstream of the transcription initiation site of the 5'-pim-1 promoter and a series of deletion mutants were ligated upstream of the chloramphenicol acetylase transferase (CAT) gene in an expression vector that was introduced into FDC/Nb2 cells, a premyeloid line that stably expresses the intermediate form of the PRLR. Analysis of PRL-treated cultures indicated that two elements [distal (DE), -427 to -336 bp and proximal (PE), - 104 to -1] but not several GAS or GAS-like sequences were required for hormone activation of the pim-1 promoter. Moreover, treatment of Nb2-11 cells with PRL activated protein binding to these elements assessed by gel mobility shift assay. Deoxyribonuclease I (DNase I) protection experiments revealed a motif containing a nuclear factor-1 (NF-1, -224 to -217 bp) half-site that was hydrolyzed when exposed to extracts from PRL-treated cells but protected by proteins from unstimulated cells. Gel mobility shift analysis of this sequence showed decreased protein binding after PRL stimulation. It is concluded that the PRLR initiates pim-1 transcription by a mechanism that involves transcriptional activation by factors that stimulate the DE- and PE-sites and derepress a NF-1-containing element. Moreover, this mechanism appears to be independent of an interaction between Stat transcription factors and GAS-like elements present within the promoter.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Regulación de la Expresión Génica/efectos de los fármacos , Prolactina/farmacología , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción , Animales , Sitios de Unión , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Eliminación de Gen , Linfoma , Mutagénesis , Factores de Transcripción NFI , Proteínas Proto-Oncogénicas c-pim-1 , Ratas , Receptores de Prolactina/efectos de los fármacos , Receptores de Prolactina/fisiología , Transducción de Señal , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Proteína 1 de Unión a la Caja YRESUMEN
This study investigates the effect of dietary carotenoids on pim-1 gene expression in mouse splenocytes. Female BALB/c mice were fed 0%, 0.02%, or 0.4% astaxanthin, beta-carotene, and lutein for two weeks. Plasma and liver were obtained for the analysis of carotenoids. Splenocytes were isolated and cultured in the presence of concanavalin A, and the level of pim-1 mRNA was determined by Northern blot analysis. None of the carotenoids were detectable in the plasma and liver of unsupplemented mice. In plasma the concentration of astaxanthin (4.9-54.7 mumol/l) was dramatically higher than that of lutein (1.4-2.0 mumol/l) and beta-carotene (0.1-0.7 mumol/l). Carotenoid uptake by the spleen but not the liver reflected that observed in plasma. In mice fed 0.4% of each carotenoid, the absolute concentration of the carotenoid in the liver was highest for astaxanthin (24 nmol/g) followed by beta-carotene (7.5 nmol/g) and lutein (1.58 nmol/g). Mice fed lutein showed a dose-related increase in pim-1 mRNA expression. The steady-state level of pim-1 mRNA in mice fed 0.4% lutein was sixfold higher than in mice fed 0.02% lutein. In contrast, dietary astaxanthin and beta-carotene did not affect pim-1 expression. Therefore, an increase in pim-1 mRNA was observed in splenocytes stimulated with concanavalin A in lutein-fed mice. This appears to be a unique effect of lutein and may be associated with its antitumor activity observed in vivo.
Asunto(s)
Anticarcinógenos/farmacología , Dieta , Luteína/farmacología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/genética , beta Caroteno/análogos & derivados , beta Caroteno/farmacología , Animales , Anticarcinógenos/metabolismo , Northern Blotting , Cromatografía Líquida de Alta Presión , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/citología , Hígado/efectos de los fármacos , Luteína/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-pim-1 , ARN Mensajero/análisis , Bazo/citología , Bazo/efectos de los fármacos , Xantófilas , beta Caroteno/metabolismoRESUMEN
Lactogen-dependent Nb2 lymphoma cells, widely employed for studying prolactin (PRL) mitogenic mechanisms, are also useful for investigations of apoptosis in T-lineage lymphocytes. Utilizing PRL-dependent Nb2-11 cultures, apoptosis-regulatory genes were evaluated for participation in dexamethasone- (DEX) provoked cell death or its inhibition by PRL. Treatment of lactogen-starved, G1-arrested Nb2-11 cells with DEX (100 nM) activated apoptosis within 12 h evaluated by flow cytometric analysis of fragmented DNA. This effect was not associated with altered expression of bcl-2, bax, or pim-1. PRL (10 ng/mL), coincubated with DEX-treated cells, completely blocked DEX-induced apoptosis. This inhibition was associated with increased expression of bcl-2 and pim-1 mRNAs, genes reported to suppress apoptosis, within 2-6 h after addition of the hormone. Moreover, the increased transcription of bcl-2 and pim-1 was coupled to increases in their protein levels. The results suggest that bcl-2, bax, and pim-1 do not play a critical role in DEX-induced apoptosis in Nb2 cells. However, expression of bcl-2, together with pim-1, may have a role in mediating the antiapoptotic actions of PRL.
Asunto(s)
Apoptosis/efectos de los fármacos , Linfoma/patología , Prolactina/farmacología , Proteínas Serina-Treonina Quinasas , Animales , Dexametasona/farmacología , Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-pim-1 , ARN Mensajero/metabolismo , Ratas , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2RESUMEN
Biologically active human interleukin-2 (IL-2) and IL-4, key lymphokines involved in immune regulation, were produced and secreted into the medium by genetically modified Nicotiana tabacum cells grown in suspension culture. Secretion through the plasma membrane and cell wall into the medium was facilitated by the natural mammalian leader sequences. IL-2 and IL-4 were detected in the medium at concentrations of 0.10 and 0.18 microgram/mL, respectively, although higher levels were detected within the lymphokine-producing cells (approximately 0.80 microgram/mL for IL-2 and approximately 0.28 microgram/mL for IL-4). By Western blot, IL-4 was found to be secreted as two small polypeptides with molecular masses of approximately 18-20 kDa. The biological activity of IL-2 was determined by cell proliferation of the IL-2-dependent murine CTLL-2 cell line, while that of IL-4 was determined by cell proliferation of the CTLL-2 cell line [CT.h4S] which was stably transfected with the human IL-4 receptor. These findings indicate that plant suspension culture can be used to produce and secrete into the medium a variety of biologically active mammalian proteins that are of clinical and diagnostic relevance.
Asunto(s)
Interleucina-2/metabolismo , Interleucina-4/metabolismo , Nicotiana/genética , Plantas Tóxicas , Secuencia de Bases , Western Blotting , Células Cultivadas , Clonación Molecular , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-2/genética , Interleucina-4/genética , Plantas Modificadas GenéticamenteRESUMEN
The in vitro effects of conjugated dienoic derivatives of linoleic acid (CLA) in combination with beta-carotene on lymphocyte and macrophage function was studied. Porcine blood lymphocytes and murine peritoneal macrophages were incubated with 0 (control), 1.78 x 10(-5), 3.57 x 10(-5) and 7.14 x 10(-5) M CLA and 0 (control), 10(-9), 10(-8) and 10(-7) M beta-carotene. CLA alone stimulated mitogen-induced lymphocyte proliferation, lymphocyte cytotoxic activity and macrophage bactericidal activity. In contrast, CLA inhibited interleukin-2 production by lymphocytes and suppressed the phagocytic activity of macrophages. beta-Carotene alone stimulated the cytotoxicity of lymphocytes and increased superoxide production by peritoneal macrophages. When present together, CLA and beta-carotene interacted in an additive manner to further enhance lymphocyte cytotoxicity and spontaneous lymphocyte proliferation. In addition, beta-carotene was able to negate the inhibitory action of CLA on the phagocytic activity of macrophages. Also, CLA and beta-carotene together seemed to suppress mitogen-induced lymphocyte proliferation. Therefore, CLA and beta-carotene; alone and in concert, act to modulate different aspects of cellular host defense.
Asunto(s)
Anticarcinógenos/farmacología , Ácidos Linoleicos/farmacología , Activación de Linfocitos/efectos de los fármacos , Macrófagos/efectos de los fármacos , beta Caroteno/farmacología , Animales , Interleucina-2/biosíntesis , Ácido Linoleico , Macrófagos/fisiología , Ratones , Superóxidos/metabolismo , PorcinosRESUMEN
The parental rat Nb2 lymphoma is a prolactin (PRL)-dependent T cell line. Exposure of a PRL-independent subline, Nb2-SFJCD1, to sodium butyrate (NaBT) causes transient reversal of their growth factor-independent proliferation in association with constitutive expression of protooncogenes pim-1 and c-myc. In the present study, we investigated the effect of NaBT treatment on the sensitivity of Nb2-SFJCD1 cells to dexamethasone (DEX)-induced apoptosis. Pretreatment with NaBT (2 mM, 72 h) partially reversed resistance to apoptosis in Nb2-SFJCD1 cells exposed to DEX (100 nM) for 12 h, assessed by flow cytometric analyses of DNA fragmentation. However, the cytolytic effect of DEX was abrogated by PRL in a time- and concentration-dependent manner. Evaluation of apoptosis-associated gene expression in NaBT-pre-treated cultures incubated with DEX or DEX+PRL indicated that the apoptosis resistance did not stem from altered bcl-2 or bax expression. However, there was a strong correlation between the resistance to DEX-activated apoptosis and their enhanced expression of pim-1 mRNA and protein. The results show that it is possible to reverse DEX-induced apoptosis of Nb2 pre-T cells and suggest the pim-1 gene product has an important role as a suppressor of this process, perhaps functioning as a mediator of PRL action.
RESUMEN
Expression of Pim-1, an oncogenic serine/threonine kinase, is highly regulated at the transcriptional, posttranscriptional, and posttranslational levels. Here, we report that expression of Pim-1 kinase is additionally regulated at the translational level. Pim-1 protein expression did not increase in Hut-78 lymphocytes in response to PMA1/ionomycin stimulation despite approximately 20-fold increases in mRNA levels, suggesting that translation was repressed. Sequence analysis of the 5'-untranslated region (UTR) indicated a long (400 nucleotide), 76% G + C-rich region, characteristics known to inhibit translation. Deletion of the 5'-UTR of pim-1 increased translation of the Pim-1 protein approximately 10-fold in vitro in reticulocyte lysates and approximately 1.6-fold in vivo in NIH-3T3 cells. When full-length 5'-UTR-containing pim-1 cDNA constructs were transfected into NIH-3T3 cells overexpressing eukaryotic translation initiation factor 4E (eIF-4E), approximately 6-fold higher levels of Pim-1 protein were produced, as compared to that produced in control NIH-3T3 cells. Moreover, eIF-4E overexpression had little effect in the absence of the 5'-UTR, suggesting that it relieved 5'-UTR-mediated inhibition of Pim-1 expression.
Asunto(s)
Conformación de Ácido Nucleico , Factores de Iniciación de Péptidos/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , ARN Mensajero/fisiología , Células 3T3 , Animales , Secuencia de Bases , Línea Celular , Factor 4E Eucariótico de Iniciación , Humanos , Metionina/metabolismo , Ratones , Datos de Secuencia Molecular , Nigericina/análogos & derivados , Nigericina/farmacología , Factores de Iniciación de Péptidos/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-pim-1 , ARN Mensajero/análisis , Reticulocitos/metabolismo , Análisis de Secuencia de ARN , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética , Transfección , beta-Galactosidasa/análisisRESUMEN
Whereas Nb2-11 lymphoma cells critically require prolactin (PRL) for growth, Nb2-SFJCD1 subline cells are growth factor independent. Treatment with the differentiating agent, sodium butyrate (NaBT), has been demonstrated previously to lead to growth arrest of Nb2-SFJCD1 cells and a transient reversion to PRL growth requirement following removal of NaBT. In the present study, the relation of NaBT-induced growth arrest to the cell cycle was examined using flow cytometry, and the effect of PRL on expression of the immediate-early proto-oncogenes, pim-1 and c-myc, in NaBT-pretreated cultures was evaluated. Treatment of Nb2-SFJCD1 cells with 2 mM NaBT for 72 h caused growth arrest in the majority of the cells in the G1 phase of the cell cycle, an effect similar to that produced by lactogen deprivation in PRL-dependent Nb2 cultures. The addition of PRL stimulated a concentration-dependent re-entry into the cell cycle. In other experiments, NaBT treatment significantly reduced the steady-state levels of pim-1 and c-myc mRNA. Stimulation with PRL induced a rapid and concentration-dependent biphasic accumulation of each mRNA with similar kinetics. Maximal expression of both proto-oncogenes occurred within 2-4 h and after 12 h. Results from mRNA stability studies suggest that the observed increases in expression of pim-1 and c-myc most likely do not reflect increased stability of the transcripts. The results indicate that NaBT-induced differentiation in autonomous Nb2-SFJCD1 causes growth arrest of the cells in the G1 phase of the cell cycle and reduces the basal levels of pim-1 and c-myc mRNAs. Mitogenic stimulation with PRL reinitiates cell cycle progression characterized by biphasic expression of each proto-oncogene. It is suggested that NaBT is a useful tool for investigation of the malignant progression from growth factor dependency to autonomy in the Nb2 lymphoma paradigm.
Asunto(s)
Butiratos/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas/genética , Animales , Northern Blotting , Ácido Butírico , División Celular/efectos de los fármacos , División Celular/fisiología , Citometría de Flujo , Fase G1/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Sustancias de Crecimiento/farmacología , Inhibidores de Histona Desacetilasas , Linfoma de Células T , Prolactina/farmacología , Proteínas Proto-Oncogénicas c-pim-1 , ARN Mensajero/metabolismo , Ratas , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimologíaRESUMEN
Bovine leukemia virus (BLV) induces a non-malignant, polyclonal, persistent lymphocytosis (PL) of circulating, CD5 B lymphocytes in cattle, with variable progression to CD5 B cell leukemia or lymphoma. We analyzed the expression of two proto-oncogenes, pim-1 and c-myc, proto-oncogenes deregulated in some human B cell leukemias and lymphomas, in peripheral blood mononuclear leukocytes (PBML) from BLV-infected PL cows. Results demonstrate that pim-1 and c-myc mRNA levels are elevated in unfractionated stimulated PBML from a sample of PL cows naturally infected with BLV. Results confirm that pim-1 is constitutively expressed, but not inducible in normal bovine peripheral blood B lymphocytes, but can be induced in the predominantly CD5 B lymphocytes from BLV-infected PL cows. Results further demonstrate that c-myc is inducible in bovine B and T lymphocytes regardless of BLV status, but the amount of induction is greater in B lymphocytes from BLV-infected PL cows than in B lymphocytes from noninfected control cows. These results suggest that pim-1 and c-myc are upregulated in B lymphocytes from BLV-infected PL cows and that deregulation of proto-oncogene expression is not limited to completely transformed cells, but can also characterize a naturally occurring, pre-neoplastic lymphocytic state.
Asunto(s)
Linfocitos B/metabolismo , Leucosis Bovina Enzoótica/genética , Regulación de la Expresión Génica , Genes myc , Linfocitosis/genética , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Animales , Bovinos , Femenino , Humanos , Interleucina-2/biosíntesis , Ionomicina/farmacología , Ratones , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-pim-1 , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Increasing the level of recovery of mammalian proteins secreted by a genetically modified Nicotiana tabacum was explored in suspension culture. As a model protein system, a mouse monoclonal antibody heavy chain gamma (MAb HC) with an antigen specificity for p-azophenylarsonate was used. Consistent with findings for other plant cell suspension culture systems expressing proteins with mammalian leader sequences, the synthesized mouse MAb HC was secreted through the plasma membrane. In addition, the majority of the MAb HC was also secreted through the cell wall into the growth medium. However, efficient recovery of the protein was only possible when the protein stabilizing agent, polyvinylpyrrolidone (PVP) was present in the plant cell growth medium. The presence of PVP increased the recovered concentration of secreted protein 35-fold from 0.010 to 0.36 micrograms protein/ml culture medium. Biological activity of the approximately 50-kDa MAb HC polypeptide was demonstrated by arsonate affinity matrix binding as determined by Western blot analysis. In addition to antigen binding activity, the secreted protein also exhibited reactivity to protein G, a protein which specifically binds mouse IgG. These findings are important because they demonstrate that culture conditions can significantly influence the concentration of a biologically active foreign protein secreted from plant cells into the media of suspension cultures. The ability to increase the efficiency of mammalian protein production in plant suspension culture systems should provide significant advantage over protein production in intact transgenic plants which require cultivation, harvesting, and expensive extraction procedures to obtain nonsecreted foreign proteins.
Asunto(s)
Anticuerpos Monoclonales/genética , Cadenas Pesadas de Inmunoglobulina/genética , Nicotiana/genética , Nicotiana/metabolismo , Plantas Tóxicas , Animales , Anticuerpos Monoclonales/inmunología , Baculoviridae/genética , Baculoviridae/metabolismo , Northern Blotting , Western Blotting , Fraccionamiento Celular , Células Cultivadas , Células Clonales/metabolismo , Regulación de la Expresión Génica/genética , Vectores Genéticos/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Ratones , Plantas Modificadas Genéticamente/genética , Povidona/farmacología , Unión Proteica , Proteínas Recombinantes/aislamiento & purificación , Transformación Genética/genética , p-Azobencenoarsonato/metabolismoRESUMEN
We have studied pim-1 proto-oncogene expression in human T cell responses to Ag receptor-generated signals. The pim-1 gene encodes a serine/threonine protein kinase that is expressed primarily in cells of hematopoietic lineage and is implicated in the intracellular signaling processes accompanying lymphocyte activation. We show here that pim-1 mRNA expression is rapidly induced after receptor cross-linking with anti-CD3 Abs. We examined the linkage of pim-1 expression to known signaling pathways generated through the T cell Ag receptor. pim-1 mRNA was not substantially induced after elevation of intracellular free Ca2+. In contrast, PMA, which directly activates PKC, induced rapid pim-1 expression. Further, anti-CD3- or PMA-induced pim-1 expression was markedly reduced by various PKC inhibitors and by deficiency of the PKC epsilon isoform in a mutant T cell line. Thus, T cell Ag receptor-linked pim-1 expression appears to be coupled to the PKC component of transmembrane signaling. Because the activation of protein kinase C has been shown to activate Raf-1 kinase activity, the involvement of Raf-1 in pim-1 expression was also investigated using a human T cell line stably transfected with an inducible Raf expression vector. Although the overexpression of activated Raf was shown to cause a substantial increase in IL-2 expression, no discernible effects on pim-1 were apparent. In addition, we examined transcriptional and post-transcriptional mechanisms involved in PKC-mediated pim-1 expression and observed that both transcriptional and post-transcriptional mechanisms are coordinately involved in the up-regulation of the pim-1 proto-oncogene.
Asunto(s)
Isoenzimas/metabolismo , Activación de Linfocitos/fisiología , Muromonab-CD3/farmacología , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/fisiología , Proto-Oncogenes , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/fisiología , Linfocitos T/metabolismo , Calcio/fisiología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/fisiología , Humanos , Ionomicina/farmacología , Leucemia-Linfoma de Células T del Adulto/patología , Proteínas Serina-Treonina Quinasas/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-pim-1 , Proteínas Proto-Oncogénicas c-raf , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Complejo Receptor-CD3 del Antígeno de Linfocito T/antagonistas & inhibidores , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Síndrome de Sézary/patología , Neoplasias Cutáneas/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
The lactogen-dependent Nb2 lymphoma line (Nb2-11) represents a useful pre-T cell model for investigation of early molecular events coupled to PRL-stimulated cell cycle progression. Expression of pim-1, a protooncogene that encodes a conserved cytosolic serine/threonine protein kinase, is rapidly induced in hematopoietic cells upon mitogen stimulation and is thought to be important for lymphocyte activation. The present study was conducted to determine whether mitogen stimulation in Nb2-11 or lactogen-independent Nb2-SFJCD1 cells provokes pim-1 gene expression. The pim-1 transcript was undetectable in control growth-arrested Nb2-11 cultures; however, PRL rapidly stimulated its expression in a biphasic manner. Peak expression occurred within 2-4 h (> 40-fold) and was followed by a second elevation at 12 h. The effect of PRL and IL-2 to induce pim-1 at 2 h was concentration dependent and not inhibited by cycloheximide. In Nb2-SFJCD1 cells, pim-1 messenger RNA was expressed in control cultures and augmented by PRL stimulation. Results from stability studies indicated that the t1/2 values for the pim-1 transcript were 79 and 81 min in PRL-stimulated Nb2-11 cells at 2 and 12 h. However, in the lactogen-treated Nb2-SFJCD1 line, it was nearly 3-fold more stable (219 min) at 2 h compared to that determined at either 12 h or in unstimulated cultures. In other experiments, PRL-stimulated expression of the pim-1 protein was evaluated in [35S]methionine-labeled cells by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In Nb2-11 cells, enhanced [35S]pim-1 expression paralleled its messenger RNA transcription through 8 h. Elevated [35S]pim-1 was detected within 1 h and peaked by 2-4 h. Therefore, pim-1 represents an immediate early gene induced by PRL stimulation in Nb2-11 cells. Its initial peak of transcription occurs early during G1 cell cycle progression, whereas a second elevation is coincident with the G1/S transition. These results demonstrate that mitogen-induced expression of pim-1 is a rapid event in Nb2 lymphoma cells and suggest that it may be associated with cell cycle progression.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-2/farmacología , Prolactina/farmacología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes/efectos de los fármacos , Animales , Ciclo Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Linfoma/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-pim-1 , Ratas , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The pim-1 gene encodes a serine/threonine protein kinase with expression restricted primarily to cells of hematopoietic lineage and is thought to play a role in the signal transduction events associated with lymphocyte activation. A rapid increase in pim-1 mRNA levels was found after stimulation of normal unseparated PBMCs with phorbol ester (PMA) and a calcium ionophore (ionomycin) with the peak level occurring 4 hr poststimulation. Treatment of PBMCs with ionomycin alone caused only a minimal increase in pim-1 mRNA, whereas treatment with PMA alone induced a large increase in pim-1 mRNA, suggesting that the activation of a signaling pathway involving protein kinase C is responsible for the accumulation of this transcript. In enriched subpopulations of resting alpha/beta-T cells, gamma/delta-T cells, and B cells, pim-1 expression was found to be constitutively expressed, albeit at lower levels in T cells. This basal level of pim-1 expression could be increased by stimulation of alpha/beta-T cells (approx fivefold) and gamma/delta-T cells (approximately sevenfold) with PMA plus ionomycin. In contrast, pim-1 expression was not inducible in B cells. In PBMCs, half-life determination studies showed that turnover of pim-1 mRNA was markedly prolonged as a result of message stabilization induced by PMA plus ionomycin treatment. In addition, stable pim-1 transcripts were also observed in all transformed lymphoid cell lines examined. Taken together, these results suggest that the stability of pim-1 transcripts may be linked to the regulation of cell growth and represent the first direct demonstration that pim-1 expression is indeed regulated in a cell-type-specific manner.