Mouse mammary tumor virus-related sequences in mouse lymphocytes are inducible by 12-O-tetradecanoyl phorbol-13-acetate

cDNA libraries from EL-4 cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) were screened for TPA-inducible sequences by differential hybridization. The most abundant inducible species was a sequence similar to that of mouse mammary tumor virus (MMTV). Induction of the mRNA corresponding to the MMTV-related sequences was already evident 30 min after TPA treatment, whereas the maximum accumulation occurred after 20 h of exposure to TPA. TPA also increased levels of MMTV-related RNA in the normal spleen cells of BALB/c and C57BL/6 mice. The level of RNA expression corresponding to MMTV-related sequences, however, was markedly elevated in EL-4 cells as compared with normal spleen cells. Southern blots of EL-4 cell DNA showed that the MMTV-related sequences were inserted into multiple locations of the EL-4 genome. Sequence analysis revealed that the MMTV-related cDNA clones included a part of the env gene and the right long terminal repeat of MMTV. However, the cDNA sequences were substantially different from published MMTV proviral sequences, most notably because of a contiguous deletion of 491 base pairs in the open reading frame within the U3 region.     

Optimization of subtractive hybridization in construction of subtractive cDNA libraries

The efficiency of subtraction, integrity of residual single-stranded cDNA, and efficient recovery of nanogram quantities of double-stranded cDNA are the three most important factors affecting quality of subtractive hybridization reactions prior to subtractive cDNA library construction. Techniques for efficient isolation of single-stranded cDNA, after subtraction, have greatly improved from early protocols based on hydroxylapatite chromatography to phenol-chloroform extraction of biotin-streptavidin-crosslinked polynucleotides or oligo(dA)-cellulose affinity chromatography. Factors affecting mRNA stability at the hybridization step, however, also have consequences that directly affect the complexity of the library and the length of cDNAs recovered. We have optimized the subtractive hybridization step in subtractive cDNA library construction to ensure that single-stranded cDNAs survive hybridization as near to full length as possible. These improvements have enabled successful construction of subtractive cDNA libraries from the nanogram quantities of single-stranded cDNA remaining after extensive liquid hybridization to high calculated C_ot values.     

Beta2-microglobulin required for cell surface expression of blastocyst MHC

Blastocyst MHC is a mouse MHC class Ib gene that is selectively expressed in blastocysts and placenta like human HLA-G, which protect fetal trophoblasts and some tumor cells from NK cell attack, and in TAP-dependent expression on the cell surface. We expressed blastocyst MHC cDNA in beta2-deficient EL-4 S3 or beta2m-transfected EL-4 S3 cells. In parental EL-4 S3 cells, only 47-kDa blastocyst MHC protein was expressed and retained in the cytoplasm. However, additional 51-kDa blastocyst MHC protein was expressed on the surface of beta2m-transfected EL-4 S3 cells. The 51-kDa protein was resistant to Endo-H, whereas the 47-kDa protein was sensitive for Endo-H. The results suggested that beta2m as well as TAP was necessary for the transportation of blastocyst MHC from endoplasmic reticulum to cell surfaces through the Golgi apparatus, similar to other MHC class I molecules.     

Purification of homogeneity and amino acid sequence analysis of a receptor protein for interleukin 1

The interleukin 1 (IL-1) receptor from mouse EL-4 thymoma cells was purified to homogeneity by a method which utilized ligand affinity chromatography and classical chromatographic techniques. After solubilization of the receptor from intact cells with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the IL-1 binding activity was purified greater than 23,000-fold. Analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot, and ligand blot demonstrated that a single protein of molecular mass of approximately 80 kDa is the IL-1 binding polypeptide. The purified protein bound IL-1 with a dissociation constant of approximately 1.1 X 10(-10) M, which is indistinguishable from the affinity of the cell-bound receptor. The amino acid composition of this protein is strikingly similar to the composition deduced from the sequence of a cDNA coding for an IL-1 receptor from EL-4 cells. Protein sequence analysis of Staphylococcus aureus V-8 protease-derived peptides yields data consistent with the sequence proposed from cloned cDNA. These studies have demonstrated that the high affinity IL-1 receptor on EL-4 cells is the 80-kDa protein.     

In vitro immunization. Effect of growth and differentiation factors on antigen-specific B cell activation and production of monoclonal antibodies to autologous antigens and weak immunogens

The antigen-specific activation of murine nonimmunized B lymphocytes subsequently used in hybridization experiments has been investigated by using phylogenetically conserved antigens or autologous immunogens. This in vitro immunization was supported by B cell growth and differentiation factors derived from phorbol myristate acetate-stimulated EL-4 thymoma cells and mixed lymphocyte cultures (MLC). A filter immuno-plaque assay was used to evaluate the effect of different activation procedures on the number of antigen-specific plaque-forming cells (PFC). We first determined the requirement for MLC-derived lymphokines in the in vitro immunization. An optimal number of antigen-specific PFC was obtained when using 33 to 50% of the supernatant from a 48-hr MLC to support the activation. B cell growth and differentiation factors derived from EL-4 cultures were then tested for their abilities to potentiate the number of PFC by using both unseparated spleen cells and highly purified Ig-positive B cells as target cells. The combination of lymphokines found in supernatants from 25% EL-4 thymoma culture and 33% MLC yielded the highest number of PFC when used to support an in vitro immunization. This optimal factor preparation was used to determine the kinetics (4 to 7 days) and the dose response (0.01 to 10 micrograms antigen/ml) of antigen-specific B cell activation before using the immunized splenocytes as parental cells in cell fusion experiments. Mouse albumin and hemoglobin, actin (25 micrograms/ml), RNA polymerase II (5 micrograms/ml), as well as syngeneic mouse serum were used to immunize BALB/c spleen cells in vitro. We obtained antigen-specific PFC by using all of the different immunogens, including syngeneic mouse serum, and the in vitro immunized cells were then used in hybridization experiments. The specific efficiencies of each fusion that made use of cells immunized with mouse albumin, hemoglobin, syngeneic mouse serum, actin, or RNA polymerase II were 12, 31, 33, 52, and 22%, respectively, which illustrated the apparent lack of immune tolerance found when the immunization was performed in culture.     

Molecular cloning of cDNA corresponding to mRNA species whose steady state levels in the thyroid are enhanced by thyrotropin. Homology of one of these sequences with ferritin H

A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.     

Detection of mRNAs present at low concentrations in rat liver by in situ hybridization: application to the study of metabolic regulation and azo dye hepatocarcinogenesis

In situ hybridization on tissue sections was used to detect mRNAs present at low concentrations during metabolic adaptation and azo dye carcinogenesis in rat liver. The method consisted of hybridizing the slices at relatively high stringency with [35S]-labeled single-stranded probes derived from cDNA insert clones into the M13 phage. L-pyruvate kinase mRNA was proved to be present at very low concentrations in hepatocytes of fasted rats and to be relatively abundant in all hepatocytes after 18 hr of refeeding on a carbohydrate-rich diet. Aldolase A mRNA concentrations have been previously shown to increase markedly in liver of 3'-methyl DAB-fed rats, with a maximum at the fourth week. We demonstrate here, using our in situ hybridization technique, that this phenomenon is not due to re-expression of this "fetal marker" in hepatocytes but to its abundancy in proliferating small cells (i.e., so-called oval and transitional cells). Small amounts were also detected in sinusoidal cells. In normal liver, aldolase A mRNAs were detected only in some sinusoidal cells.     

EL-4 tumor cell-induced human and rabbit platelet aggregations

EL-4 tumor cells were assayed in vitro for their ability to aggregate two kinds of platelets. An inhibition study showed that the EL-4 tumor cell can induce platelet aggregation by at least two different mechanisms. One, mediated by thrombin, was dominant with rabbit platelets because hirudin, which specifically inhibits thrombin, considerably suppressed the rabbit platelet aggregation induced by EL-4 tumor cells. In contrast, EL-4 cells induced the aggregation of human platelets even in citrated PRP. It is the apyrase-sensitive pathway that is believed to work in human platelets. The human platelet responses to EL-4 tumor cells clearly differed from those of rabbit platelets in terms of inhibition by hirudin and apyrase and of reactivity in citrated PRP. Both phospholipase A2 and dibutyryl cAMP strongly inhibited EL-4 tumor cell-induced platelet aggregation in both rabbit and human platelets. These two compounds may block a vital step in platelet aggregation that is elicited by the EL-4 tumor cells. Our results show that human platelet response to tumor cells is not necessarily deducible from experimental data obtained with animal platelets.     

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.     

转录因子Foxo3a高表达对小鼠T淋巴瘤EL-4细胞周期和凋亡的影响

为了研究转录因子Foxo3a高表达对小鼠T淋巴瘤EL-4细胞周期和凋亡的影响,采用电穿孔法将真核表达载体pEGFP-N1/Foxo3a转染小鼠T淋巴瘤细胞系EL-4细胞,并通过聚合酶链式反应和免疫印迹法分别检测Foxo3a mRNA及蛋白表达。转录因子Foxo3a高表达后,采用细胞计数法绘制其细胞生长曲线;采用荧光显微镜法及流式细胞仪定性和定量观察典型EL-4细胞凋亡形态特征、细胞凋亡百分率及细胞周期变化情况。结果表明,转录因子Foxo3a真核表达质粒pEGFP-N1/Foxo3a经酶切鉴定及测序检测序列正确。转染pEGFP-N1/Foxo3a的小鼠EL-4细胞表达Foxo3a mRNA和蛋白水平显著升高。Foxo3a高表达明显抑制EL-4细胞的增殖能力,并使EL-4细胞发生明显G2期阻滞(P<0.001)。Foxo3a基因高表达后,荧光显微镜可以观察到典型凋亡的细胞形态。同时,EL-4细胞凋亡百分率显著升高(P<0.01)。结果提示,Foxo3a高表达可以有效抑制小鼠T淋巴瘤细胞体外细胞增殖,使细胞周期G2时相阻滞,并具有诱导细胞凋亡的作用。     

A murine T lymphocyte antigen belongs to a supergene family of type II integral membrane proteins

A murine cell surface, disulfide-linked 85kDa dimer, defined with murine mAb A1, is expressed at high levels on EL-4 cells, but at low levels on normal C57BL/6 T cells. A similar structure is recognized by the rat mAbs YE1/32 and YE1/48. We isolated a cDNA clone encoding the antigen recognized by mAb A1 by immunoselection of a cDNA library in the eukaryotic expression vector CDM8. COS 7.2 cells transfected with this cDNA clone expressed an mAb A1-reactive 85 kDa disulfide-linked dimer with 44 kDa subunits, which was also reactive with the mAbs YE1/32 and YE1/48. The A1 gene displayed extensive strain polymorphism, underwent no rearrangement in EL-4, and hybridized with multiple restriction fragments, suggesting that it is a member of a multi-gene family. The deduced polypeptide contained 262 residues with an m.w. of 30,648, multiple cysteines, and three potential N-linked glycosylation sites, consistent with previous observations. In contrast to most integral membrane proteins, the putative A1 protein had features of a type II integral membrane protein structure, with its carboxyl terminus exposed extracellularly and an intracytoplasmic amino terminus. There was significant homology with several type II integral membrane proteins, including the human and chicken asialoglycoprotein receptors, and especially the human low affinity Fc epsilon receptor, in the putative extracellular domains of these proteins. This analysis suggested that the A1 gene belongs to a novel supergene family of type II integral membrane proteins and suggested that the A1 protein itself may be involved in binding a soluble ligand such as carbohydrates or immunoglobulin.     

Cloning of mRNA nucleotide sequences amplified during DNA replication in the regenerating liver

A library of double-stranded cDNA has been constructed using the mRNA of regenerating rat liver 20 hr after partial hepatectomy. The differential screening of the library with the regenerating liver specific and the resting-liver-specific single-stranded cDNA probes has identified 11 cDNA clones which sequences are preferentially expressed in regenerating rat liver. The RNA dot blot hybridization has shown that levels of RNA complementary to these clones are 3 to 8-fold higher in dividing cells as compared with resting cells.     

Soluble factor-independent stimulation of human B cell response by mouse thymoma cells. Cyclosporine A-resistant and -sensitive cell contact signals

In a Th cell-dependent antibody response, the Th act on B cells partly via a helper activity that is cell contact-dependent and cyclosporine A (CsA)-resistant. This activity seems to be required to induce responsiveness of the B cells toward T cell-derived soluble factors (cytokines) generally believed to be essential for B cell proliferation as well as for Ig secretion. In our study, we have investigated a system in which human B cells are stimulated by mutant EL-4 thymoma cells of mouse origin. It was found that human B cells proliferate and secrete Ig (either 1) in the presence of EL-4 cells plus human T cell supernatant (T-SUP), or 2) in the presence of EL-4 cells alone which have been induced with PMA or IL-1. The first situation conformed to the known synergy between CsA-resistant Th signal and cytokines. However, the B response due to PMA-induced EL-4 cells was special. The PMA-inducible helper activity was CsA-sensitive at the same CsA concentration that inhibited IL-2 secretion of EL-4 cells, but the murine factors in EL-4 supernatant had no effect on human B cells; the helper effect did not occur across a semipermeable membrane. Any contribution of soluble factors from contaminating human T cells was ruled out by adding single human B cells by flow microfluorimetry to cultures with EL-4 cells and PMA. Such B cells generated clonal IgM, IgG, and/or IgA responses. CsA, thus, interfered with some cell contact-mediated signal. However, CsA did not reduce the amount of LFA-1 molecules on EL-4 cells. In conclusion, EL-4 cells can induce proliferation and differentiation of human B cells in a soluble factor-independent manner, via CsA-resistant and CsA-sensitive helper activities. This may represent an alternative pathway of B cell activation.     

Macrophage activation to kill Leishmania tropica: characterization of a T cell-derived factor that suppresses lymphokine-induced intracellular destruction of amastigotes

Factors obtained from phorbol myristate acetate (PMA)-stimulated EL-4 thymoma cells, a continuous T cell line, suppressed lymphokine-induced macrophage activation to kill intracellular Leishmania tropica amastigotes. Suppression of this macrophage effector activity was dependent upon concentration of EL-4 fluids admixed with lymphokines in infected macrophage cultures, and was not due to residual PMA or factors released from unstimulated EL-4 cells. Fluids from PMA-stimulated EL-4 cells did not affect the expression of microbicidal activity by macrophages activated in vivo as a consequence of infections with Mycobacterium bovis strain BCG, nor did they abrogate intracellular killing activities by C3H/HeJ macrophages primed by BCG infection and triggered by lymphokines in vitro. That the action of this EL-4 suppressor activity was at the priming stage of macrophage activation was confirmed by kinetic studies: EL-4 fluids added to lymphokine-treated cells in the first 4 hr of treatment completely suppressed intracellular killing of L. tropica; fluids added after 4 hr were not effective. The effects of these EL-4 factors appeared to be selective: of three effector activities of activated macrophages tested, induction of resistance to infection, tumor cytotoxicity, and intracellular destruction of L. tropica, only intracellular killing by lymphokine-treated macrophages was significantly suppressed. These T cell-derived soluble suppressor factor(s) may provide insight into mechanisms of immunosuppression during leishmanial disease and perhaps other intracellular parasitic infections.     

Mutant EL-4 thymoma cells polyclonally activate murine and human B cells via direct cell interaction

In this report we show that three mutagenized sublines of (murine) EL-4 thymoma cells can constitutively activate human and/or murine B cells via an MHC-nonrestricted cell-cell interaction. The activation signal is not by itself mitogenic but renders B cells capable of proliferating in response to interleukin 2 (IL 2). In addition, one of the mutant EL-4 sublines can constitutively respond by release of IL 2 in the presence of IL 1-containing macrophage (P388D1) supernatant. The exact relationships between these functional properties of the mutant EL-4 thymoma cells and those associated with activated normal T helper-cells remain to be established. However, the EL-4 cells provide a unique system to study in parallel murine and human B cell responses. In particular, the following observations were made during the present study. First, anti-Ig antibodies (anti-Ig) were required for B cell activation in conjunction with two EL-4 sublines acting only on murine B cells, whereas with a third subline acting on both murine and human B cells, anti-Ig was not required. Anti-Ig by itself did not lead to significant B cell activation in the absence of mutant EL-4 (or normal T) cells. Second, the growth factor-stimulated proliferation of EL-4-activated B cells, following separation of the B cells from the EL-4 cells, lasted only 2 days. These results, thus, indicate that the requirement for a surface Ig-mediated B cell activation signal depends on the quality/intensity of a direct T cell signal and that cell-cell interactions may exert a more stringent control over the growth factor responsiveness of B cells as compared with T cells.     

Identification of sequences within the murine granulocyte-macrophage colony-stimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells

Phorbol esters (TPA) and concanavalin A (ConA) are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF) production in murine thymoma EL-4 cells by mRNA stabilization. The role of the 3'-untranslated region (3'-UTR) in GM-CSF mRNA stabilization induced by TPA and ConA in EL-4 cells was examined by transfection studies using chloramphenicol acetyltransferase (CAT) constructions. The GM-CSF 3'-UTR contains a 63-nucleotide region at its 3' end with repeating ATTTA motifs which is responsible for mRNA degradation in a variety of cell types (Shaw, G., and Kamen, R. (1986) Cell 46, 659-666). We produced constructs containing most of the GM-CSF 3'-UTR (303 nucleotides, pRSV-CATgm) or the 3'-terminal AT-rich region (116 nucleotides, pRSV-CATau) and measured CAT enzyme activity and CAT mRNA after transient transfection into EL-4 and NIH 3T3 cells. Low levels of CAT activity were seen in both cells with either plasmid compared with levels of CAT activity obtained with pRSV-CAT. TPA treatment caused an approximately 10-fold increase in CAT activity and mRNA in EL-4 cells transfected with pRSV-CATgm. No increases were seen in EL-4 cells transfected with pRSV-CATau or pRSV-CAT. No response to TPA was detected in transfected NIH 3T3 cells, indicating that the response to TPA is relatively cell-specific. There was no increase in CAT activity after ConA treatment in EL-4 or NIH 3T3 cells transfected with any of the constructs suggesting that the GM-CSF 3'-UTR lacks elements that can respond alone to ConA. Nuclear run-on and actinomycin D chase experiments in EL-4 cells showed that TPA induces CAT activity via mRNA stabilization. By linker-substitution mutagenesis we show that TPA inducibility depends on a 60-nucleotide region of the 3'-UTR whose 5' end is located 160 nucleotides upstream of the 5' end of the AU-rich region.     

In vivo natural antitumor resistance against murine EL-4 lymphoma cells in lethally irradiated syngeneic C57Bl/6 mice

Natural resistance has been detected in lethally irradiated C57Bl/6 (B6) mice inoculated intravenously with the ascites form of a syngeneic B6 leukemia. EL-4 cells were injected into lethally irradiated (800 R) B6 mice and tumor cell proliferation was evaluated by 125IUdR uptake in different organs 4 days after the challenge. Differential growth of lymphoma cells was observed when young mice were injected as compared with older mice and when mice were treated with agents known to interfere with natural resistance (e.g., poly(I:C), FLV-P, carrageenan, cyclophosphamide, high doses of irradiated cells). Similar results were obtained by measuring rapid clearance of 125IUdR-labeled EL-4 cells from lungs of intact B6 mice. In vivo cold competition studies, employing EL-4 and several other tumor lines of the same or different haplotype, showed that only EL-4 and RBL-5 cells were capable of inhibiting syngeneic resistance against EL-4 tumor. On the contrary, YAC-1 lymphoma cells, the most susceptible target to natural killer-mediated cytotoxicity in vitro, did not compete. These results suggest that EL-4 cells express membrane determinants not detectable on normal H-2b parental bone marrow cells and are susceptible to natural resistance against hemopoietic tumor cells in lethally irradiated syngeneic B6 mice.