RESUMEN
Interaction of the translational repressor 4E-BP1 with the mRNA cap binding protein eIF4E plays an important role in the regulation of translation initiation. This interaction is modulated by phosphorylation of 4E-BP1 on at least six residues. However, analysis of the functional importance of the individual phosphorylation sites is complicated by the lack of information about the kinases and phosphatases involved in modulating phosphorylation of each site. The goal of the present study was to establish a system whereby alterations in the interaction of 4E-BP1 with eIF4E could be easily and directly measured. In initial studies, both eIF4E and 4E-BP1 were expressed as recombinant proteins coupled to variants of green fluorescent protein (ECFP and EYFP, respectively). Addition of purified EYFP--4E-BP1 to ECFP--eIF4E caused both a decrease in emission intensity at 480 nm and an increase at 535 nm indicating that protein-protein interaction had occurred. The interaction was stoichiometric and was blocked by eIF4G. Phosphorylation of EYFP--4E-BP1 by the mitogen-activated protein kinase ERK2, but not by casein kinase CK-II, also attenuated the interaction. Results using proteins in which the fluorescent protein tag was located at either the N- or C-terminus suggested that, in the protein complex, the N-termini of the two proteins are in close spatial proximity, as are the C-termini. Overall, the results demonstrate that fluorescence resonance energy transfer between EYFP--4E-BP1 and ECFP--eIF4E is a valuable tool in directly measuring alterations in the interaction of the two proteins.
Asunto(s)
Proteínas Portadoras/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Sitios de Unión , Proteínas Portadoras/química , Quinasa de la Caseína II , Transferencia de Energía , Factor 4E Eucariótico de Iniciación , Factor 4G Eucariótico de Iniciación , Colorantes Fluorescentes/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Factores de Iniciación de Péptidos/química , Fosfoproteínas/química , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de FluorescenciaRESUMEN
The alpha-subunit of eukaryotic initiation factor eIF2 is a preferred substrate for the double-stranded RNA-activated protein kinase, PKR. Phosphorylation of eIF2alpha converts the factor from a substrate into a competitive inhibitor of the guanine nucleotide exchange factor, eIF2B, leading to a decline in mRNA translation. Early studies provided evidence implicating PKR as the kinase that phosphorylates eIF2alpha under conditions of cell stress such as the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum, i.e., the unfolded protein response (UPR). However, the recent identification of a trans-microsomal membrane eIF2alpha kinase, termed PEK or PERK, suggests that this kinase, and not PKR, might be the kinase that is activated by misfolded protein accumulation. Similarly, genetic studies in yeast provide compelling evidence that a kinase termed GCN2 phosphorylates eIF2alpha in response to amino acid deprivation. However, no direct evidence showing activation of the mammalian homologue of GCN2 by amino acid deprivation has been reported. In the present study, we find that in fibroblasts treated with agents that promote the UPR, protein synthesis is inhibited as a result of a decrease in eIF2B activity. Furthermore, the reduction in eIF2B activity is associated with enhanced phosphorylation of eIF2alpha. Importantly, the magnitude of the change in each parameter is identical in wildtype cells and in fibroblasts containing a chromosomal deletion in the PKR gene (PKR-KO cells). In a similar manner, we find that during amino acid deprivation the inhibition of protein synthesis and extent of increase in eIF2alpha phosphorylation are identical in wildtype and PKR-KO cells. Overall, the results show that PKR is not required for increased eIF2alpha phosphorylation or inhibition of protein synthesis under conditions promoting the UPR or in response to amino acid deprivation.
Asunto(s)
Aminoácidos/metabolismo , Señalización del Calcio/fisiología , Retículo Endoplásmico/fisiología , Fibroblastos/fisiología , Iniciación de la Cadena Peptídica Traduccional , ARN Bicatenario/metabolismo , eIF-2 Quinasa/metabolismo , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Células Cultivadas , Ácido Egtácico/farmacología , Embrión de Mamíferos , Exones , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Hidroquinonas/farmacología , Ratones , Ratones Noqueados , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , ARN Bicatenario/genética , Eliminación de Secuencia , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/genéticaRESUMEN
Regulation of translation of mRNAs coding for specific proteins plays an important role in controlling cell growth, differentiation, and transformation. Two proteins have been implicated in the regulation of specific mRNA translation: eukaryotic initiation factor eIF4E and ribosomal protein S6. Increased phosphorylation of eIF4E as well as its overexpression are associated with stimulation of translation of mRNAs with highly structured 5'-untranslated regions. Similarly, phosphorylation of S6 results in preferential translation of mRNAs containing an oligopyrimidine tract at the 5'-end of the message. In the present study, leucine stimulated phosphorylation of the eIF4E-binding protein, 4E-BP1, in L6 myoblasts, resulting in dissociation of eIF4E from the inactive eIF4E.4E-BP1 complex. The increased availability of eIF4E was associated with a 1.6-fold elevation in ornithine decarboxylase relative to global protein synthesis. Leucine also stimulated phosphorylation of the ribosomal protein S6 kinase, p70(S6k), resulting in increased phosphorylation of S6. Hyperphosphorylation of S6 was associated with a 4-fold increase in synthesis of elongation factor eEF1A. Rapamycin, an inhibitor of the protein kinase mTOR, prevented all of the leucine-induced effects. Thus, leucine acting through an mTOR-dependent pathway stimulates the translation of specific mRNAs both by increasing the availability of eIF4E and by stimulating phosphorylation of S6.
Asunto(s)
Leucina/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Biosíntesis de Proteínas , Proteínas Quinasas , ARN Mensajero/genética , Proteínas Ribosómicas/metabolismo , Línea Celular , Factor 4E Eucariótico de Iniciación , Músculos/citología , Músculos/metabolismo , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/metabolismo , Fosforilación , Proteína S6 Ribosómica , Serina-Treonina Quinasas TORRESUMEN
The present study was designed to investigate the mechanism through which leucine and histidine regulate translation initiation in L6 myoblasts. The results show that both amino acids stimulate initiation and coordinately regulate the activity of eukaryotic initiation factor eIF2B. The changes in eIF2B activity could be explained in part by modulation of the phosphorylation state of the alpha-subunit of eIF2. The activity changes might also be a result of modulation of the phosphorylation state of the eIF2B epsilon-subunit, because deprivation of either amino acid caused a decrease in eIF2Bepsilon kinase activity. Leucine, but not histidine, additionally caused a redistribution of eIF4E from the inactive eIF4E.4E-BP1 complex to the active eIF4E.eIF4G complex. The redistribution was a result of increased phosphorylation of 4E-BP1. The changes in 4E-BP1 phosphorylation and eIF4E redistribution associated with leucine deprivation were not observed in the presence of insulin. However, the leucine- and histidine-induced alterations in global protein synthesis and eIF2B activity were maintained in the presence of the hormone. Overall, the results suggest that both leucine and histidine regulate global protein synthesis through modulation of eIF2B activity. Furthermore, under the conditions employed herein, alterations in eIF4E availability are not rate-controlling for global protein synthesis but might be necessary for regulation of translation of specific mRNAs.
Asunto(s)
Aminoácidos/farmacología , Proteínas Portadoras , Proteínas Musculares/biosíntesis , Músculo Esquelético/metabolismo , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Factores de Iniciación de Péptidos/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación , Histidina/farmacología , Leucina/deficiencia , Leucina/farmacología , Músculo Esquelético/citología , Fosfoproteínas/metabolismo , FosforilaciónRESUMEN
The alpha-subunit of eukaryotic initiation factor eIF2 (eIF2alpha) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulated in vivo by phosphorylation of eIF2alpha at Ser51. In the present study, rat eIF2alpha was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to >90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2alpha kinase activity and was rapidly phosphorylated by the eIF2alpha kinases HCR and PKR. A variant of eIF2alpha in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2alpha was not phosphorylated by either HCR or PKR, demonstrating that the kinases specifically phosphorylate the correct site in the recombinant protein even in the absence of the other two subunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2alpha has been developed. Use of the wildtype and variant forms of eIF2alpha to measure eIF2alpha kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions.
Asunto(s)
Factor 2 Eucariótico de Iniciación/aislamiento & purificación , Regulación de la Expresión Génica/genética , Animales , Baculoviridae/fisiología , Secuencia de Bases , Línea Celular , Cromatografía de Afinidad , Cartilla de ADN/química , Electroforesis en Gel de Poliacrilamida , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Hemo/farmacología , Fosforilación , Reacción en Cadena de la Polimerasa , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Represoras/efectos de los fármacos , Proteínas Represoras/metabolismo , Spodoptera/citología , Spodoptera/virología , Factores de Tiempo , eIF-2 Quinasa/metabolismoRESUMEN
Modulation of protein/protein interaction is an important mechanism involved in regulation of translation initiation. Specifically, regulation of the interaction of eIF2 with the guanine nucleotide exchange factor, eIF2B, is a key mechanism for controlling translation under a variety of conditions. Phosphorylation of the alpha-subunit of eIF2 converts the protein into a competitive inhibitor of eIF2B by causing an increase in the binding affinity of eIF2B for eIF2. Consequently, it has been assumed that the alpha-subunit of eIF2 is directly involved in binding to eIF2B. In the present study, eIF2 was found to bind only to the delta- and epsilon-subunits of eIF2B, and eIF2B was shown to bind only to the beta-subunit of eIF2 by far-Western blot analysis. The binding site on eIF2beta for either the eIF2B holoprotein, or the isolated delta- or epsilon-subunits of eIF2B was shown to be located within approximately 70 amino acids of the C terminus of the protein. Phosphorylation of the alpha-subunit of eIF2 did not promote binding of eIF2B to the isolated subunit. However, it did cause an increase in the affinity of eIF2B for eIF2. Finally, phosphorylation by protein kinase A of the beta-subunit of eIF2 in the C-terminal portion of the protein increased the guanine nucleotide exchange activity of eIF2B, whereas phosphorylation by casein kinase II or protein kinase C was without effect.
Asunto(s)
Factor 2 Eucariótico de Iniciación/química , Iniciación de la Cadena Peptídica Traduccional , Proteínas/química , Animales , Sitios de Unión , Quinasa de la Caseína II , Factor 2B Eucariótico de Iniciación , Factores de Intercambio de Guanina Nucleótido , Mutagénesis , Fosforilación , Unión Proteica , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , RatasRESUMEN
The phosphorylation states of three proteins implicated in the action of insulin on translation were investigated, i.e., 70-kDa ribosomal protein S6 kinase (p70S6k), eukaryotic initiation factor (eIF) 4E, and the eIF-4E binding protein 4E-BP1. Addition of insulin caused a stimulation of protein synthesis in L6 myoblasts in culture, an effect that was blocked by inhibitors of phosphatidylinositide-3-OH kinase (wortmannin), p70S6k (rapamycin), and mitogen-activated protein kinase (MAP kinase) kinase (PD-98059). The stimulation of protein synthesis was accompanied by increased phosphorylation of p70S6k, an effect that was blocked by rapamycin and wortmannin but not PD-98059. Insulin caused dephosphorylation of eIF-4E, an effect that appeared to be mediated by the p70S6k pathway. Insulin also stimulated phosphorylation of 4E-BP1 as well as dissociation of the 4E-BP1.eIF-4E complex. Both rapamycin and wortmannin completely blocked the insulin-induced changes in 4E-BP1 phosphorylation and association of 4E-BP1 and eIF-4E; PD-98059 had no effect on either parameter. Finally, insulin stimulated formation of the active eIF-4G.eIF-4E complex, an effect that was not prevented by any of the inhibitors. Overall, the results suggest that insulin stimulates protein synthesis in L6 myoblasts in part through utilization of both the p70S6k and MAP kinase signal transduction pathways.
Asunto(s)
Factores Eucarióticos de Iniciación , Insulina/farmacología , Músculo Esquelético/fisiología , Biosíntesis de Proteínas/fisiología , Transducción de Señal/fisiología , Androstadienos/farmacología , Animales , Proteínas Portadoras/metabolismo , Inhibidores Enzimáticos/farmacología , Factor 4E Eucariótico de Iniciación , Flavonoides/farmacología , Músculo Esquelético/efectos de los fármacos , Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Polienos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Represoras/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus , WortmaninaRESUMEN
Protein synthesis is inhibited in both rat liver and isolated rat hepatocytes following deprivation of single essential amino acids. The aim of the present study was to define the time course of changes in peptide-chain initiation, albumin synthesis, and albumin mRNA following histidine deprivation and the reversal of these changes in response to readdition of the deprived amino acid. A further aim was to ascertain whether there was an accommodation of the inhibition of initiation following long-term amino acid deprivation. Primary cultures of rat hepatocytes were maintained in serum-free medium containing either all amino acids (complete medium) or all except histidine. Synthesis of total protein was reduced to 34% of control values following 48 hr of histidine deprivation and was restored to control values within 1 hr of addition of complete medium to histidine-deprived cells. These changes in protein synthesis were due to translational regulation involving initiation. No accommodation of the inhibition was observed following long-term deprivation of histidine as has been observed under other conditions of cellular stress. The synthesis of albumin was reduced to a greater extent than that of total protein, and required 72 hr to recover to control values following return to complete medium. These changes in albumin synthesis were due to a combination of altered initiation and a mechanism involving pretranslational regulation as evidenced by corresponding alterations in albumin mRNA. The results show that amino acid availability controls protein synthesis in liver cells through both translational and pretranslational mechanisms.
Asunto(s)
Albúminas/biosíntesis , Histidina/deficiencia , Hígado/metabolismo , Iniciación de la Cadena Peptídica Traduccional/fisiología , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Albúminas/genética , Albúminas/metabolismo , Animales , Células Cultivadas , Hígado/citología , RatasRESUMEN
When primary cultures of rat hepatocytes were placed in a chemically defined serum-free medium containing a combination of insulin, glucagon, and dexamethasone, the synthesis of albumin and total protein and the cellular content of RNA and DNA were maintained at constant values for 8 days. Despite the constant rate of albumin synthesis, secretion of the protein increased more than twofold during the initial 4 days in culture and was then maintained at a value similar to that observed in vivo through day 8. This observation suggested an initial defect in albumin secretion that was corrected with time in culture. Deprivation of insulin between days 2 and 5 resulted in a decline in albumin secretion to approximately 40% of the control value. The decline in albumin secretion was accompanied by proportional decreases in albumin synthesis, albumin mRNA, and albumin gene transcription. Return of insulin-deprived cells to complete medium on day 5 restored albumin synthesis and secretion as well as albumin mRNA to control values by day 8. Deprivation of either glucagon or dexamethasone also resulted in reduced albumin synthesis and secretion accompanied by proportional decreases in albumin mRNA and gene transcription. However, the magnitude of the changes in these parameters was less with glucagon or dexamethasone deprivation compared with insulin deprivation. Return of glucagon- or dexamethasone-deprived cells to complete medium on day 5 restored albumin synthesis and secretion as well as albumin mRNA to control values by day 8.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Dexametasona/farmacología , Expresión Génica/efectos de los fármacos , Glucagón/farmacología , Insulina/farmacología , Hígado/fisiología , Albúmina Sérica/genética , Animales , Células Cultivadas , Hígado/citología , ARN Mensajero/metabolismo , Ratas , Albúmina Sérica/biosíntesisRESUMEN
The studies presented herein were designed to investigate the role of insulin in maintaining the steady-state number of ribosomes in primary cultures of rat hepatocytes. The RNA content of hepatocytes maintained in the presence of insulin did not change during the 7 days of culture. In contrast, the RNA content declined significantly following 2 days of insulin deprivation and after 4 days without insulin reached a new steady-state value that was approximately 60% of that observed for hepatocytes maintained in the presence of the hormone. Following addition of insulin, the RNA content of insulin-deprived hepatocytes started to increase within 6 h and was restored to the control value within 48 h. The amounts of 18 and 28 S RNA, and thus the number of ribosomes, changed in concert with the total RNA content. Furthermore, synthesis of total cellular protein responded in parallel to the changes in RNA content, suggesting that the number of ribosomes was the primary determinant of protein synthesis under the conditions of these experiments. About one-half of the increase in RNA content following the addition of insulin to insulin-deprived cells could be accounted for by a reduction in the rate of degradation of ribosomes. The remainder of the change in RNA content must have resulted from an increase in ribosome biogenesis. This possibility was confirmed by measuring [3H]uridine incorporation into ribosomal RNA present in mature ribosomes. The rate of synthesis of ribosomal RNA was reduced in parallel with the fall in RNA content in insulin-deprived hepatocytes and was restored to the control value within 3 h of the addition of insulin. Alterations in the synthesis of ribosomal RNA in response to insulin deprivation and replacement were associated with parallel changes in transcription of rDNA as measured in nuclear run-on assays using a DNA probe corresponding to the external transcribed spacer region of rat rDNA. The data demonstrate that insulin regulates the number of ribosomes in primary cultures of rat hepatocytes by accelerating the rate of transcription of rDNA and by slowing the rate of ribosome degradation.
Asunto(s)
ADN Ribosómico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Insulina/farmacología , Hígado/metabolismo , ARN Ribosómico/biosíntesis , Ribosomas/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Células Cultivadas , ADN/metabolismo , Cinética , Leucina/metabolismo , Hígado/efectos de los fármacos , Biosíntesis de Proteínas , Ratas , Ribosomas/efectos de los fármacos , Ribosomas/ultraestructura , Tritio , Uridina/metabolismo , Uridina Trifosfato/metabolismoRESUMEN
Carbonic anhydrase (CA) was examined in two adipocyte cell lines, 3T3-L1 and 3T3-F442A. Both CA III and non-CA III activities, measured by 18O mass spectrometry, were present in 3T3-L1 and 3T3-F442A adipocytes; however, no CA activity was detected in 3T3 preadipocytes of either line. These observations were supported by immunoblot experiments employing CA III and CA II isoform-specific antisera. CA III, a major protein in rodent and murine adipocytes, and CA II, another isoform known to be present in adipose tissue, were observed only in the differentiated 3T3 adipocytes. The differentiation-dependent expression of these isozymes may imply an adipocyte-related role for CA. Compared with cultures maintained in the absence of insulin, 3T3 adipocytes maintained in the presence of insulin exhibited 65-90% lower concentrations of CA III. CA II was unaffected. This negative effect of insulin on CA III may explain the metabolic regulation of adipose CA III observed in vivo. After media changes, 3T3 adipocyte cultures rapidly lower media pH, which in turn lowers the bicarbonate/CO2 of bicarbonate/CO2-buffered media. Cultures maintained at low pH displayed 50-90% lower concentrations of CA II and CA III. Similarly, cultures maintained in a low bicarbonate/CO2 media (GibCO2-I medium containing 1 mM bicarbonate under an atmosphere of 100% humidified air) displayed 30-50% lower CA II and CA III concentrations. Thus CA II and CA III concentrations are influenced by pH and bicarbonate/CO2. Neither effect, the pH or the GibCO2-I media effect, was associated with changes in the concentration of pyruvate carboxylase or ATP citrate lyase (2 markers of adipocyte differentiation). Because the regulation by pH and bicarbonate/CO2 may be relatively selective for CA in adipocytes, a simple method for reducing the concentration/activity of CA in 3T3 adipocytes is described that may be a useful tool for studies on the physiological role of the enzyme.
Asunto(s)
Tejido Adiposo/enzimología , Anhidrasas Carbónicas/metabolismo , Células 3T3 , Tejido Adiposo/citología , Secuencia de Aminoácidos , Animales , Bicarbonatos/farmacología , Dióxido de Carbono/farmacología , Diferenciación Celular/fisiología , Medios de Cultivo , Concentración de Iones de Hidrógeno , Insulina/farmacología , Ratones , Datos de Secuencia Molecular , Concentración OsmolarRESUMEN
DNAs of cell lines derived from human metastatic tumors were transfected into tumorigenic, nonmetastasizing murine cells (BALB 3T12-3) to determine if the capacity to metastasize could be conferred by this transfer of portions of the human genome. Using the calcium phosphate method, whole cell DNA was cotransfected into the murine cells along with a neomycin-resistance gene. Recipient murine cells (10(6] which grew in neomycin, indicating successful transfection, were injected via tail vein into 4- to 6-week-old male athymic nude mice. Animals were sacrificed if they appeared ill or at times up to 24 weeks after injection if they remained healthy. Murine cells transfected with DNA from one cell line derived from a hepatic metastasis of a human pancreatic adenocarcinoma (NCI-ZRY) formed experimental pulmonary metastases in 11 of 13 animals injected. Neither murine cells (unmanipulated BALB 3T12-3 cells) nor murine cells transfected with DNA from the same cell type (BALB 3T12-3 cells transfected with BALB 3T12-3 DNA) produced experimental metastases when each cell type was injected into 20 and 10 animals, respectively (P2 less than 0.0001). The results are consistent with the hypothesis that expression of a structural or regulatory protein encoded on human DNA conferred the metastatic phenotype to the recipient murine cells.
Asunto(s)
ADN de Neoplasias , Ratones/genética , Metástasis de la Neoplasia/genética , Transfección , Animales , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Masculino , Ratones Desnudos , Fenotipo , Células Tumorales CultivadasRESUMEN
An antiproliferative suppressor lymphokine was produced from rat T-cells specifically in response to the poorly immunogenic syngeneic mammary adenocarcinoma 13762A. The tumor-induced suppressor lymphokine (TISL) was produced late in culture (peak production on Days 4 and 5) and showed strong but selective inhibitory activity on a variety of immune responses. The immune peritoneal exudate cell response to a highly immunogenic clone from the parental tumor (clone 18A) and the concanavalin A-stimulated response of nonimmune spleen cells were inhibited strongly by TISL. In contrast, the immune spleen cell response to 13762A and the lipopolysaccharide response of nonimmume spleen cells were unaffected. Preliminary molecular weight and physicochemical analysis of TISL indicated that the molecule was large (Mr greater than 350,000); partially sensitive to 75 degrees C treatment for 15 min and to pH 2.0 treatment; only partly degraded by the enzymes trypsin, chymotrypsin, and proteinase K; and completely destroyed by boiling. Although TISL was produced specifically in response to 13762A tumor, prior immunization in vivo was not necessary for the induction of the suppressor lymphokine. These results indicate that populations of rat lymphocytes contain naturally occurring TISL secreting cells, which can be activated specifically by tumor antigens such as those expressed by 13762A.
Asunto(s)
Adenocarcinoma/inmunología , Citotoxicidad Inmunológica , Neoplasias Mamarias Experimentales/inmunología , Factores Supresores Inmunológicos/biosíntesis , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos/inmunología , Proteínas del Sistema Complemento/inmunología , Femenino , Activación de Linfocitos , Ratas , Ratas Endogámicas F344RESUMEN
We examined the in vitro responses of immune lymphocytes to the tumor antigens of the syngeneic rat mammary adenocarcinoma 13762A. This tumor readily metastasizes to lymph node and lungs and is poorly immunogenic. Rats were immunized with a highly immunogenic clone (18A) which was isolated as a spontaneous variant from the parental 13762A tumor. Clone 18A grew progressively in irradiated rats but regressed completely in normal rats. Animals immune to 18A tumor were also immune to parental 13762A. Lymphocytes obtained from the spleen and peritoneum of immune rats were tested for specific proliferation to parental 13762A tumor and clone 18A to determine whether similar cross-reactivity to these tumors occurred in vitro. We found an anatomical difference in localization of immune lymphocytes which reacted to the two tumor cell lines. Immune peritoneal exudate cells (PEC) responded strongly to clone 18A but poorly to 13762A, while immune spleen cells from the same animals responded predominantly to 13762A tumor. After 7 days culture, PEC proliferating in response to clone 18A contained 84-95% W3/25+ T-helper cells, and only 5-8% OX8+ cytotoxic/suppressor cells, while analogous cultures of spleen cells responding to parental 13762A tumor consisted of 60-80% W3/25+ cells and 20-23% OX8+ cells. Immune spleen cell cultures stimulated with 13762A tumor generated cytotoxic lymphocytes which specifically lysed both parental 13762A and clone 18A cells. We conclude that despite cross-reactivity in vivo and in vitro, antigens present on 13762A and 18A tumor cells stimulated different subsets of immune T cells.