ABSTRACT
Flow-PRA is a flow cytometric method for both anti-HLA class I and class II antibody (Ab) detection. We evaluated this technique for Ab screening in patients awaiting kidney transplantation. After having established a rigorous threshold for positivity, a three-dilution difference in sensitivity between Flow-PRA and complement-dependent cytotoxicity (CDC) persisted. The sensitivity of the method was satisfactory since all CDC-positive sera were also found to be positive with the Flow-PRA method. Discrimination between anti-HLA class I and class II Abs was excellent. Furthermore, all sera responsible for a positive flow cytometry crossmatch (FCXM) and a negative CDC-crossmatch (CDCXM) at the time of a putative transplant were found to be positive with Flow-PRA beads. The specificity was excellent for anti-class I Ab detection since no false positive serum was found. On the other hand, the specificity was lower for anti-class II detection, since 8.3% (2/24) false positive results were detected among all the negative sera tested. Overall, our results suggested that Flow-PRA should be of value for anti-HLA Ab screening prior to kidney transplantation.
Subject(s)
Flow Cytometry , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class I/immunology , Isoantibodies/blood , Kidney Transplantation , Histocompatibility Testing , Humans , Male , Reproducibility of ResultsSubject(s)
HLA-D Antigens/immunology , Isoantibodies/blood , Kidney Transplantation/immunology , Antibody-Dependent Cell Cytotoxicity , B-Lymphocytes/immunology , Creatinine/blood , Graft Rejection/immunology , Graft Survival/immunology , Histocompatibility Testing , Humans , Kidney Transplantation/physiology , Postoperative Complications/immunology , Reoperation , T-Lymphocytes/immunology , Treatment FailureABSTRACT
The formation of the pre-B cell receptor (BCR) corresponds to an important checkpoint in B cell development that selects pro-B (pre-BI) cells expressing a functionally rearranged immunoglobulin mu (Igmu) heavy chain protein to undergo the transition to the pre-B (pre-BII) cell stage. The pre-BCR contains, in addition to Igmu, the surrogate light chains lambda5 and VpreB and the signal transducing proteins Igalpha and Igbeta. The absence of one of these pre-BCR components is known to arrest B cell development at the pre-BI cell stage. Disruption of the Pax5 gene, which codes for the B cell-specific activator protein (BSAP), also blocks adult B lymphopoiesis at the pre-BI cell stage. Moreover, expression of the mb-1 (Igalpha) gene and VH-to-DHJH recombination at the IgH locus are reduced in Pax5-deficient B lymphocytes approximately 10- and approximately 50-fold, respectively. Here we demonstrate that complementation of these deficiencies in pre-BCR components by expression of functionally rearranged Ig mu and chimeric Igmu-Igbeta transgenes fails to advance B cell development to the pre-BII cell stage in Pax5 (-/-) mice in contrast to RAG2 (-/-) mice. Furthermore, the pre-BCR is stably expressed on cultured pre-BI cells from Igmu transgenic, Pax5-deficient bone marrow, but is unable to elicit its normal signaling responses. In addition, the early developmental block is unlikely to be caused by the absence of a survival signal, as it could not be rescued by expression of a bcl2 transgene in Pax5-deficient pre-BI cells. Together, these data demonstrate that the absence of Pax5 arrests adult B lymphopoiesis at an early developmental stage that is unresponsive to pre-BCR signaling.
Subject(s)
B-Lymphocytes/physiology , DNA-Binding Proteins/physiology , Hematopoietic Stem Cells/physiology , Leukopoiesis/physiology , Nuclear Proteins/physiology , Receptors, Antigen, B-Cell/physiology , Transcription Factors , Animals , B-Lymphocytes/cytology , Cells, Cultured , DNA-Binding Proteins/genetics , Hematopoietic Stem Cells/cytology , Humans , Immunoglobulin mu-Chains/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Nuclear Proteins/genetics , PAX5 Transcription Factor , Signal TransductionABSTRACT
Pax-5 codes for the transcription factor BSAP which is expressed throughout B cell development except in terminally differentiated plasma cells. Gene targeting experiments in the mouse revealed a differential dependency of fetal and adult B-lymphopoiesis on this transcription factor. BSAP is required for B-lineage commitment in the fetal liver and for progression beyond an early pro-B cell stage in adult bone marrow. The characterization of Pax-5-deficient pro-B cells demonstrated an important role of BSAP in the regulation of the CD19, mb-1 (Ig alpha) and N-myc genes as well as in the developmental pathway controlling VH-to-DHJH recombination at the immunoglobulin heavy-chain (IgH) locus. The human PAX-5 gene was recently shown to participate together with the IgH locus in the chromosomal translocation t(9;14)(p13;q32). This translocation is characteristic of a small subset of non-Hodgkin lymphomas exhibiting plasmacytoid differentiation. The translocated PAX-5 gene is deregulated by the insertion of IgH regulatory elements into its 5' region, which may contribute to tumorigenesis by interfering with the shut-down of PAX-5 transcription and thus with the completion of plasma cell differentiation.
Subject(s)
B-Lymphocytes/cytology , DNA-Binding Proteins/physiology , Leukopoiesis/physiology , Nuclear Proteins/physiology , Transcription Factors/physiology , Adult , Animals , DNA-Binding Proteins/genetics , Down-Regulation , Gene Expression Regulation , Gene Expression Regulation, Neoplastic , Gene Rearrangement, B-Lymphocyte , Genes, Immunoglobulin , Humans , Immunoglobulin Heavy Chains/genetics , Liver/embryology , Lymphoma, Non-Hodgkin/genetics , Mice , Mutation , Nuclear Proteins/genetics , PAX5 Transcription Factor , Transcription Factors/genetics , Translocation, GeneticABSTRACT
CD20 is a B-lineage-specific gene expressed at the pre-B-cell stage of B-cell development that disappears on differentiation to plasma cells. As such, it serves as an excellent paradigm for the study of lineage and developmental stage-specific gene expression. Using in vivo footprinting we identified two sites in the promoter at -45 and -160 that were occupied only in CD20+ B cells. The -45 site is an E box that binds basic helix-loop-helix-zipper proteins whereas the -160 site is a composite PU.1 and Pip binding site. Transfection studies with reporter constructs and various expression vectors verified the importance of these sites. The composite PU.1 and Pip site likely accounts for both lineage and stage-specific expression of CD20 whereas the CD20 E box binding proteins enhance overall promoter activity and may link the promoter to a distant enhancer.
Subject(s)
Antigens, CD20/genetics , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Cell Lineage/genetics , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Antigens, CD20/immunology , Base Sequence , Binding Sites , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Lineage/immunology , Helix-Loop-Helix Motifs , Humans , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Tumor Cells, CulturedABSTRACT
Pax-5 codes for the transcription factor BSAP which is expressed in all B-lymphoid tissues in addition to the developing central nervous system and testis. Within the B-lymphoid lineage, Pax-5 expression is already detected in the earliest B cell progenitors and persists up to the mature B cell stage. Targeted inactivation of the Pax-5 gene in the mouse germline revealed a differential dependency of fetal and adult B-lymphopoiesis on this transcription factor. Pax-5 is required for the differentiation of the earliest B-lineage-committed precursor cells in the fetal liver. In contrast, B cell development in the adult bone marrow progresses up to an early pro-B cell stage in the absence of Pax-5 function. The expression of CD19, Ig alpha (mb-1) and N-myc is severely reduced in Pax-5-deficient pro-B cells. These BSAP target genes are, however, unlikely to explain the early developmental block based on their known function in B cell development. Moreover, VH-to-DHJH rearrangements at the immunoglobulin heavy-chain locus are approximately 50-fold reduced in Pax-5-deficient pro B-cells, while the DH-to-JH rearrangements occur at a normal frequency. However, the expression of rearranged mu heavy-chain transgenes does not allow Pax-5-deficient pro-B cells to develop further to the pre-B cell stage. Together these data demonstrate therefore that B cell development in the Pax-5 deficient bone marrow is arrested at an early pro-B cell stage which is not yet responsive to pre-B cell receptor signaling.
Subject(s)
B-Lymphocytes/cytology , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors , Animals , Cell Differentiation , DNA-Binding Proteins/genetics , Gene Expression Regulation , Hematopoietic Stem Cells , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Joining Region/genetics , Immunoglobulin Variable Region/genetics , Mice , Nuclear Proteins/genetics , PAX5 Transcription FactorABSTRACT
The gene defective in X-linked agammaglobulinemia (XLA) encodes a novel protein kinase termed Bruton's tyrosine kinase (Btk). Whereas the XLA phenotype is confined to abnormalities of B-cell development and function, Btk is expressed not only in B-lymphocyte lineage but also in myeloid lineage cells. The first 450 basepairs of the Btk promoter fused to a luciferase gene displayed a similar cell-type specificity. Critical binding sites for the transcription factors PU.1 and Sp1 were identified in the proximal portion of the Btk promoter upstream of a cluster of transcriptional start sites. Mutation of either the PU.1 or Sp1 site markedly reduced the activity of a Btk promoter-luciferase reporter construct in transfection experiments. In addition, PU.1 directly transactivated the Btk promoter, and deletion of the PU.1 binding site abolished this effect. This study implicates PU.1 and Sp1 as major regulators of Btk expression and provides a foundation for further study of the regulation of this gene in XLA patients that lack Btk mRNA.
Subject(s)
DNA-Binding Proteins/metabolism , Neoplasm Proteins/genetics , Promoter Regions, Genetic , Protein-Tyrosine Kinases/genetics , Sp1 Transcription Factor/metabolism , Agammaglobulinaemia Tyrosine Kinase , Animals , Base Sequence , Binding Sites , Cell Line, Transformed , Chlorocebus aethiops , Enzyme Induction , Gene Expression Regulation, Leukemic , Gene Expression Regulation, Neoplastic , Genes, Reporter , HeLa Cells , Humans , Leukemia/pathology , Lymphoma, B-Cell/pathology , Molecular Sequence Data , Plasmacytoma/pathology , Protein-Tyrosine Kinases/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Retroviridae Proteins, Oncogenic , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
The CD19, CD20 and CD22 genes encode transmembrane proteins that are of vital importance to B-cell function. Similar to the immunoglobulin (Ig) genes, they are expressed in a lineage-specific and developmentally regulated manner. Here, John Kehrl and colleagues describe how an understanding of the transcriptional regulation of the CD19, CD20 and CD22 genes is leading to valuable insights into some of the important molecular events that occur in B-cell development and differentiation.
Subject(s)
Antigens, CD/biosynthesis , Antigens, Differentiation, B-Lymphocyte/biosynthesis , B-Lymphocytes/immunology , Cell Adhesion Molecules , Cell Differentiation/immunology , Gene Expression Regulation/immunology , Lectins , Animals , Antigens, CD/genetics , Antigens, CD19 , Antigens, CD20 , Antigens, Differentiation, B-Lymphocyte/genetics , Promoter Regions, Genetic/genetics , Sialic Acid Binding Ig-like Lectin 2ABSTRACT
The CD20(B1) gene encodes a B cell-specific protein involved in the regulation of human B cell proliferation and differentiation. Studies with 5' deletion CD20 promoter-CAT constructs have previously revealed two regions of the promoter between bases -186 and -280 and between bases -280 and -454 which contained positive regulatory elements. In this study we identified a sequence element present in the most proximal region located between bases -214 and -201, TTCTTCTAATTAA, which is important in the high constitutive expression of CD20 in mature B cells and the induction of CD20 in pre-B cells. This sequence element was referred to as the BAT box and its deletion significantly reduced the activity of a CD20 promoter-CAT construct in B cells. Mobility shift assays with various mutant probes and B cell nuclear extracts demonstrated that the core sequence TAAT was essential for binding to this site. Cross competition experiments with an octamer sequence from the Ig heavy chain promoter, the BAT box, and a TA-rich sequence present in the CD21 promoter revealed that all three sequences bound the same nuclear proteins suggesting that the BAT box binding proteins were Oct-1 and Oct-2. Southwestern blotting and UV cross-linking studies confirmed that the BAT box binding proteins were Oct-1 and Oct-2. The affinity of the BAT box binding proteins for the BAT box was approximately 25-fold less than for the octamer sequence and the BAT box binding proteins dissociated from the BAT box 10-fold more rapidly than from the octamer sequence. Despite this lower affinity, a trimer of the BAT box sequence was as efficiently transactivated by an Oct-2 expression vector as was a trimer of the octamer sequence in HeLa cells. The BAT box and Oct-2 were also implicated in the induction of CD20 in the pre-B cell line, PB-697, via phorbol esters. The induction of CD20 mRNA was temporally associated with induction of Oct-2 mRNA and a BAT box-deleted CD20-CAT construct, in contrast to the wild type, was poorly induced by phorbol esters. Together these results suggest that the BAT box binding proteins are important in the B cell specific expression of CD20 and perhaps CD21.
Subject(s)
Antigens, CD/genetics , Antigens, Differentiation, B-Lymphocyte/genetics , B-Lymphocytes/metabolism , DNA-Binding Proteins/genetics , Promoter Regions, Genetic , Antigens, CD/metabolism , Antigens, CD20 , Antigens, Differentiation, B-Lymphocyte/metabolism , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Base Sequence , Binding Sites , Cell Line , DNA/metabolism , DNA Mutational Analysis , DNA-Binding Proteins/metabolism , HeLa Cells , Host Cell Factor C1 , Humans , Molecular Sequence Data , Mutation , Octamer Transcription Factor-1 , Octamer Transcription Factor-2 , Receptors, Complement 3d/genetics , Sequence Deletion , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factors/metabolism , Transcriptional Activation , Tumor Cells, CulturedABSTRACT
Treatment of human B lymphocytes with an optimal concentration of okadaic acid, an inhibitor of phosphatases 1 and 2A, resulted in the induction of the transcription factor, AP-1 and a marked increase in NF-kappa B levels. In contrast, no effect on the levels of the octamer binding proteins, Oct-1 or Oct-2, were found. Since both AP-1 and NF-kappa B have been reported to be important in the induction of the tumor necrosis factor-alpha (TNF-alpha) gene we examined the effects of okadaic acid on TNF-alpha mRNA levels. Treatment with okadaic acid resulted in a striking increase in TNF-alpha mRNA transcripts within 1 h of stimulation and large amounts of TNF-alpha were released into the culture media. Although okadaic acid provides a potent inductive signal for AP-1 and NF-kappa B it did not induce either B cell proliferation or immunoglobulin secretion.
Subject(s)
B-Lymphocytes/metabolism , Ethers, Cyclic/pharmacology , NF-kappa B/biosynthesis , Proto-Oncogene Proteins c-jun/biosynthesis , Tumor Necrosis Factor-alpha/biosynthesis , B-Lymphocytes/drug effects , Cell Division , Cells, Cultured , Cycloheximide/pharmacology , Genes, fos/genetics , Genes, jun/genetics , Humans , Immunoglobulins/metabolism , Okadaic Acid , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/geneticsABSTRACT
A diverged homeobox gene, HB24, which is known to be induced following lymphocyte activation, was introduced into Jurkat T cells under the control of a constitutive promoter. Stable transfectants of HB24 were established that expressed high levels of HB24 mRNA and possessed an altered phenotype suggestive of activated T cells. A number of genes known to be induced following T cell activation and associated with cell growth were increased in the transfectants, including c-fos, c-myc, c-myb, HLA-DR, lck, NF-kappa B, interleukin-2 and interleukin-2 receptor alpha (IL-2R alpha). Analysis of IL-2R alpha expression by transient transfection of IL-2R alpha promoter constructs into the HB24 transfectants revealed constitutive expression (about 60% of phytohemagglutinin- and phorbol ester-activated Jurkat cells) that was dependent on the kappa B site in the IL-2R alpha promoter. Furthermore, as a consequence of the increased HB24 mRNA levels, the Jurkat HB24 transfectants proliferated more rapidly than control cell lines. Thus, stable expression of HB24 confers an activation phenotype on a human T cell line, implicating this gene as an important transcriptional factor during T cell activation and growth.
Subject(s)
Genes, Homeobox , Lymphocyte Activation , T-Lymphocytes/metabolism , Base Sequence , Cell Cycle , Cell Division/genetics , Cell Line , Gene Expression , Genes, fos , Genes, jun , Genes, myc , Humans , Lymphocyte Specific Protein Tyrosine Kinase p56(lck) , Molecular Sequence Data , NF-kappa B/genetics , Plasmids , Promoter Regions, Genetic , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , RNA, Messenger/metabolism , Receptors, Interleukin-2/genetics , T-Lymphocytes/immunology , Thymidine/metabolism , TransfectionSubject(s)
Antigens, CD/genetics , Antigens, Differentiation, B-Lymphocyte/genetics , B-Lymphocytes/immunology , Antigens, CD20 , Base Sequence , Binding Sites/genetics , Cell Line , Chromosome Mapping , DNA/genetics , Gene Expression , HeLa Cells , Humans , Infant, Newborn , Molecular Sequence Data , Promoter Regions, Genetic , Sequence Deletion , T-Lymphocytes/immunology , Transcriptional ActivationABSTRACT
A genomic clone spanning a large portion of the 5' untranscribed region of the CD20 gene was isolated. Deletion analysis of subcloned fragments identified several regulatory elements. A major positive cis-acting element was localized between base pairs -290/-186. A second positive regulatory element was localized between -454/-280 and negative regulatory elements were present in the region between bp -828/-454. The sequence -280/-186 conferred B cell-specific expression on a heterologous, TATA box containing c-fos promoter. Electrophoretic mobility shift assays with overlapping oligonucleotide probes spanning -280/-186 revealed that a 25-bp probe (-225/-201) bound a nuclear protein present in B cell lines expressing the CD20/B1 antigen but not in Jurkat (T cell), U937 (promonocytic), U251 (glioma), or HeLa cells. To confirm the functional significance of this sequence, a trimer of this region was subcloned into the c-fos promoter containing CAT plasmid. Expression was observed only in BJA-B and HS-Sultan cells but not in CD20/B1- cell lines. This sequence element is also important in phorbol ester-induced CD20 expression in the pre-B cell line BP-697. These results partially characterize several regulatory elements present in the CD20 promoter that are likely important in the B cell-specific expression of the CD20 gene.
Subject(s)
Antigens, CD/genetics , Antigens, Differentiation, B-Lymphocyte/genetics , B-Lymphocytes/physiology , Promoter Regions, Genetic , Antigens, CD20 , DNA Mutational Analysis , DNA-Binding Proteins/physiology , Enhancer Elements, Genetic , Gene Expression Regulation/drug effects , Genes , Humans , Oligonucleotides/chemistry , Restriction Mapping , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Transforming growth factor-beta (TGF-beta) inhibits B cell Ig secretion and reduces B cell membrane Ig expression. The addition of TGF-beta to human B lymphocyte cultures stimulated with Staphylococcus aureus Cowan strain I and IL-2 completely inhibited B cell Ig secretion (greater than 90%) and decreased B cell surface IgM, IgD, kappa L chain, and lambda L chain expression. In contrast, TGF-beta had only minimal effects on two other B cell membrane proteins, HLA-DR and CD20. Internal labeling with [35S]methionine and immunoprecipitation with anti-IgM, anti-kappa, and anti-lambda antibodies revealed a striking reduction in kappa L chain in the presence of TGF-beta. A less pronounced reduction in lambda L chain and microH chain was also noted. Northern blot analysis of RNA purified from B cells treated with TGF-beta for varying time intervals revealed a significant decrease in steady state kappa and lambda L chain mRNA levels. Furthermore, a significant decrease in the switch from the membrane forms of mu and gamma to their respective secreted forms was noted in the presence of TGF-beta. Nuclear run-on experiments demonstrated decreased transcription of kappa L chain. The effects of TGF-beta on two transcriptional regulatory factors, Oct-2 and nuclear factor (NF) kappa B, known to be important in Ig gene transcription were examined. Oct-2 mRNA levels and both Oct-2 and NF-kappa B proteins in nuclear extracts were not altered by treatment with TGF-beta. In contrast, levels of the transcriptional factor AP-1, which is not known to be important in B cell Ig production, were reduced by TGF-beta. These findings demonstrate that TGF-beta decreases B lymphocyte Ig secretion by inhibiting the synthesis of Ig mRNA and inhibiting the switch from the membrane form to the secreted forms of mu and gamma mRNA. The mechanism by which TGF-beta inhibits Ig chain synthesis is unclear although it does not involve inhibition of the binding of NF-kappa B or Oct-2 to their respective target sequences.
Subject(s)
B-Lymphocytes/drug effects , Immunoglobulins/biosynthesis , RNA, Messenger/analysis , Transforming Growth Factor beta/pharmacology , B-Lymphocytes/immunology , Cells, Cultured , DNA-Binding Proteins/analysis , Genes, Immunoglobulin , Humans , Interleukin-6/analysis , Interleukin-6/biosynthesis , Interleukin-6/pharmacology , NF-kappa B/analysis , Octamer Transcription Factor-2 , Proto-Oncogene Proteins c-jun , Transcription Factors/analysis , Transcription, GeneticABSTRACT
To examine the role of protein phosphatases in T cell activation, Jurkat cells were treated with okadaic acid, an inhibitor of type 1 and 2A phosphatases, and nuclear extracts were examined for the presence of AP1 as a measure of early T cell activation. Okadaic acid was found to be a potent inducer of AP1. In contrast to phorbol esters such as phorbol myristate acetate (PMA), the induction of AP1 by okadaic acid occurs predominantly by transcriptional activation of the jun and fos family of proto-oncogenes. Surprisingly, while the addition of phytohemagglutinin further enhanced the induction of AP1, the addition of PMA inhibited it. Okadaic acid treatment was found to dramatically increase mRNA transcripts of the jun family of proto-oncogenes including c-jun, junD, and junB and to a lesser extent the fos family including c-fos and fra-1. By comparison, PMA is a very inefficient inducer of the jun gene family in Jurkat cells. Similar to its effect on the induction of AP1 by okadaic acid, PMA inhibits the induction of c-jun mRNA by okadaic acid. Transfection of c-jun promoter constructs confirmed the marked difference between PMA and okadaic acid in inducing c-jun transcription. The induction of AP1 by okadaic acid suggests that protein phosphatases 1 and 2A (PP1 and PP2A) may be involved in T cell activation as important negative regulators of the transcription factor AP1.
Subject(s)
DNA-Binding Proteins/biosynthesis , Ethers, Cyclic/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Proto-Oncogene Proteins/biosynthesis , T-Lymphocytes/drug effects , Transcription Factors/biosynthesis , Base Sequence , Chloramphenicol O-Acetyltransferase/genetics , Humans , Molecular Sequence Data , Okadaic Acid , Phosphorylation , Promoter Regions, Genetic , Protein Kinases/metabolism , Proto-Oncogene Proteins c-jun , RNA, Messenger/genetics , Transcription, Genetic , TransfectionABSTRACT
We have used a specific phosphatase inhibitor, okadaic acid, to examine the role of two phosphatases, PP1 and PP2A, in the induction of NF-kappa B and the long terminal repeat of the human immunodeficiency virus type 1 (HIV-LTR). Treatment of Jurkat cells with okadaic acid induced NF-kappa B in nuclear extracts. The rate of induction by okadaic acid was delayed compared to the induction of NF-kappa B by phorbol myristate acetate (PMA). The induction of NF-kappa B by okadaic acid was enhanced by cycloheximide or phytohemagglutinin (PHA). In contrast to PMA, okadaic acid appeared to induce NF-kappa B independently of protein kinase C (PKC). That the NF-kappa B induced by okadaic acid was functional was demonstrated by the marked increase in CAT activity that occurred in Jurkat, BJA-B, and U251 cells that were transfected with HIV-LTR-CAT and treated with okadaic acid. The increase in CAT activity triggered by okadaic acid was dependent on the presence of the NF-kappa B sites in the long terminal repeat of HIV as assessed by deletion and mutation analysis. Similarly to its effect on the induction of NF-kappa B, PHA added together with okadaic acid resulted in a further increase in CAT activity. Somewhat surprisingly, the addition of PMA inhibited the increase in CAT activity in response to okadaic acid, which suggests that the activation of PKC may also induce inhibitory factors.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Ethers, Cyclic/pharmacology , HIV Long Terminal Repeat/drug effects , NF-kappa B/biosynthesis , Cell Line , Gene Expression Regulation/drug effects , Humans , Okadaic Acid , Phosphoprotein Phosphatases/antagonists & inhibitors , Phytohemagglutinins/pharmacology , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
We examined the effect of the human T lymphotropic virus type 1 (HTLV-I) Tax gene product on the human transforming growth factor beta 1 (TGF-beta 1) promoter. Transfection of deleted constructs of the TGF-beta 1 promoter revealed regions homologous with AP-1 binding sites that were required for Tax-induced transactivation of the TGF-beta 1 promoter. In addition, we examined the expression and secretion of TGF-beta in fresh leukemic cells isolated from patients with adult T cell leukemia (ATL) and in HTLV-1-infected T cell lines. We report that fresh leukemic cells from ATL patients constitutively produce high levels of TGF-beta 1 mRNA and secrete TGF-beta 1 but not TGF-beta 2 into the culture medium. In addition, long-term ATL cell lines expressed significant amounts of TGF-beta 1 mRNA as well as detectable levels of TGF-beta 1 protein. These results suggest a role for Tax in the upregulation of TGF-beta 1 in HTLV-I-infected cells.