Soluble dipeptidyl peptidase IV (CD-26) in serum of patients with colorectal carcinoma.

Dipeptidyl peptidase IV(DPPIV) or CD26 is a widely expressed ectopeptidase with functions in immune response such as the activation of T lymphocytes. It is expressed in the colon mucosal epithelium only when this becomes malignant. The aim of our study is to ascertain the soluble DPPIV/CD26 levels in the serum of patients with colorectal carcinoma, compared with the levels in healthy individuals. From an accrual of 70 healthy individuals and 99 patients diagnosed with colorectal adenocarcinoma, the levels of soluble DPPIV/CD26 were defined by colored enzymatic spectrophotometric response on the Gly-Pro-Paranitroaniline substrate. The patients diagnosed with colorectal cancer have a higher CD26 level than healthy subjects (p<0.05). Of these patients, those with metastatic colorectal disease have a significantly higher soluble CD26 level (p<0.01). It has also been found that patients with high LDH (lactatodeshidrogenase) and CEA (carcinoembrionary antigen) levels show higher CD26 levels (p<0.01) than patients with normal levels of such carcinoma markers.

Cellular redox state and activating protein-1 are involved in ascorbate effect on calcitriol-induced differentiation.

Ascorbate has been related to the differentiation of several mesenchymal cells including haematopoietic cells. We have previously demonstrated that ascorbate enhances the activity of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) on monocytic differentiation of HL-60 cells. Here, we show that ascorbate-mediated modification of cellular redox state and AP-1 (activating protein-1) DNA binding during early phases are related to the enhancing effect of ascorbate on differentiation. Ascorbate, but not its fully oxidized form, dehydroascorbate, or an ascorbate analogue with a low rate of oxidation, ascorbate-2-phosphate, enhanced the differentiation induced by 1 alpha,25(OH)2D3, modified cytosolic reactive oxygen species levels and mitochondrial redox potential (delta psi m), and modulated AP-1 DNA binding in HL-60 cells. Ascorbate itself increased AP-1 binding to DNA in noninduced cells, whereas it inhibited AP-1 binding in 1 alpha,25(OH)2D3-induced cells. However, ascorbate increased the mRNA levels of c-jun, junB, and c-fos in 1 alpha,25(OH)2D3-induced cells. Taken together, these results suggest that the enhancing effect of ascorbate on HL-60 differentiation induced by 1 alpha, 25(OH)2D3 is related to its effect on the cellular redox state and the modulation of AP-1 activity.

Extracellular HIV type 1 Tat protein induces CD69 expression through NF-kappaB activation: possible correlation with cell surface Tat-binding proteins.

The HIV-1 Tat protein, essential for HIV-1 gene expression and viral replication, is known to be secreted by infected cells and has pleiotropic effects on various cell functions. It seems that extracellular Tat may exert its functions on cellular targets by at least two different mechanisms, namely, by adsorptive endocytosis, and by a possible interaction with cell surface receptor(s). Here we report that extracellular Tat activates AIM/CD69 gene transcription through an NF-kappaB-dependent pathway in the erythroleukemia cell line K562. Tat induces NF-kappaB binding to DNA as a result of IkappaBalpha phosphorylation and degradation, which depend on the intracellular redox state. We found that the second Tat-coding exon is required for CD69 gene trans-activation, but not for HIV LTR gene transcription. Fluorescein-labeled Tat proteins were used to study cell surface binding sites and cellular uptake of the proteins. Full-length Tat protein has specific binding sites on the surface of K562 cells, whereas truncated Tat1-48, which is efficiently internalized by the cells, does not bind to the cell surface. Our results suggest that extracellular Tat may activate a cell surface-mediated pathway that induces intracellular signal transduction in K562 cells, leading to the activation of NF-kappaB and the transcription of NF-kappaB-dependent genes, such as CD69.

Induction of apoptosis by vanilloid compounds does not require de novo gene transcription and activator protein 1 activity.

The vanilloid compounds, capsaicin and resiniferatoxin, are quinone analogues that inhibit the NADH-plasma membrane electron transport system and induce apoptosis in transformed cells. Because disruption of the mitochondrial transmembrane potential (deltapsi(m)) is a common metabolic alteration in all apoptotic processes, we have evaluated the role of mitochondrial permeability transition in apoptosis induced by vanilloids in Jurkat cells. Using a cytofluorimetric approach, we have determined that DNA nuclear loss induced by vanilloids is preceded by an increase of the production of reactive oxygen species (ROS) and by a subsequent deltapsi(m) dissipation in T-cell lines. Overexpression of Bcl-2 and pretreatment with either the immunosuppressant cyclosporin A or the glutathione precursor N-acetyl-L-cysteine blocked deltapsi(m) disruption and apoptosis, but not the generation of ROS induced by these compounds. Capsaicin and resiniferatoxin were found to activate both isoforms of c-jun-NH2-kinase (JNK), with a maximal activity after 30 min of treatment. Despite the activation of JNK, there was no induction of activator protein 1 (AP-1) activity as determined by gel shift assay or of induction of an AP-1-responsive reporter. On the other hand, vanilloids did not signal for c-Raf kinase and extracellular signal-regulated kinases 1 and 2. We suggest that ROS generation by inhibition of the NADH-dependent plasma membrane electron transport system resulted in the oxidation of mitochondrial megachannel pores that allows for the disruption of deltapsi(m) and apoptosis, and that AP-1 activation is not required for vanilloid-induced apoptosis.

Role of ascorbate in the activation of NF-kappaB by tumour necrosis factor-alpha in T-cells.

The first product of ascorbate oxidation, the ascorbate free radical (AFR), acts in biological systems mainly as an oxidant, and through its role in the plasma membrane redox system exerts different effects on the cell. We have investigated the role of ascorbate, AFR and dehydroascorbate (DHA) in the activation of the NF-kappaB transcription factor in Jurkat T-cells stimulated by tumour necrosis factor-alpha (TNF-alpha). Here we show, by electrophoretic mobility shift assays, that ascorbate increases the binding of NF-kappaB to DNA in TNF-alpha-stimulated Jurkat cells. The ability of ascorbate to enhance cytoplasmic inhibitory IkBalpha protein degradation correlates completely with its capacity to induce NF-kappaB binding to DNA and to potentiate NF-kappaB-mediated transactivation of the HIV-1 long terminal repeat promoter in TNF-alpha-stimulated Jurkat cells but not in cells stimulated with PMA plus ionomycin. AFR behaves like ascorbate, while DHA and ascorbate phosphate do not affect TNF-alpha-mediated NF-kappaB activation. These results provide new evidence for a possible relationship between the activation of the electron-transport system at the plasma membrane by ascorbate or its free radical and redox-dependent gene transcription in T-cells.

Transcriptional regulation of the gene encoding the human C-type lectin leukocyte receptor AIM/CD69 and functional characterization of its tumor necrosis factor-alpha-responsive elements.

The human activation antigen CD69 is a member of the C-type animal lectin superfamily that functions as a signal-transmitting receptor. Although the expression of CD69 can be induced in vitro on cells of most hematopoietic lineages with a wide variety of stimuli, in vivo it is mainly expressed by T-lymphocytes located in the inflammatory infiltrates of several human diseases. To elucidate the mechanisms that regulate the constitutive and inducible expression of CD69 by leukocytes, we isolated the promoter region of the CD69 gene and carried out its functional characterization. Sequence analysis of the 5'-flanking region of the CD69 gene revealed the presence of a potential TATA element 30 base pairs upstream of the major transcription initiation site and several putative binding sequences for inducible transcription factors (NF-kappa B, Egr-1, AP-1), which might mediate the inducible expression of this gene. Transient expression of CD69 promoter-based reporter gene constructs in K562 cells indicated that the proximal promoter region spanning positions -78 to +16 contained the cis-acting sequences necessary for basal and phorbol 12-myristate 13-acetate-inducible transcription of the CD69 gene. Removal of the upstream sequences located between positions -78 and -38 resulted in decreased promoter strength and abolished the response to phorbol 12-myristate 13-acetate. We also found that tumor necrosis factor-alpha (TNF-alpha) is capable of inducing the surface expression of the CD69 molecule as well as the promoter activity of fusion plasmids that contain 5'-flanking sequences of the CD69 gene, suggesting that this cytokine may regulate in vivo the expression of CD69.(ABSTRACT TRUNCATED AT 250 WORDS)

Human herpesvirus-6 and the course of human immunodeficiency virus infection.

The aim of this study is to investigate the relationship between human herpesvirus type 6 (HHV-6) and cytomegalovirus (CMV) infection and progression of AIDS disease. A group of 52 HIV-1-seropositive patients was examined for HHV-6 DNA expression in peripheral blood mononuclear cells and for CMV DNA in serum. We found that 21.1% (n = 52) and 12% (n = 25) of them tested positive for HHV-6 and CMV DNA, respectively. In contrast, only 3.3% (n = 29) and 0% (n = 29) of control healthy HIV-1-seronegative donors tested positive for HHV-6 and CMV, respectively. In light of these results, the possible role of HHV-6 as a cofactor in AIDS development has also been assessed by closely following, over 6 years, the course of an HIV-1-seropositive person who had a dramatic loss in the total number of CD4+ cells along with a spontaneous production of HIV-1 p24 antigen in vitro and who also showed progression to AIDS when coinfected with HHV-6. These observations have spurred our prospective analysis of the possible clinical significance of coinfection with HHV-6 and HIV.

Selective decrease of CD26 expression in T cells from HIV-1-infected individuals.

The decrease of CD4+ cells in AIDS patients is widely documented, although the selective loss within different subsets of CD4+ cells and the mechanisms involved in this phenomenon are controversial. In the present report we have analyzed the proliferative response to Ag and mitogen of peripheral blood T lymphocytes from HIV-infected individuals, the phenotype profile of CD26+ and CD26- subset of cells and their infectivity by the HIV. The expression of CD26 Ag, either in CD4+ or CD8+ cells, was clearly diminished in all the patients tested. On the other hand, the expression of CD29 seems not to be affected, nevertheless T cells from these patients were unable to generate a proliferative response against soluble Ag. In 11 out of 13 patients, polymerase chain reaction studies demonstrated that the CD26- subset of CD4+ cells was the main reservoir for HIV-1 in infected individuals and HIV-1 virus preferentially infected in vitro CD4+/CD26- subpopulation. This capacity for preferential infectivity, together with the selective loss of cells expressing CD26 Ag, helps to explain the progressive impairment in the immune system of these patients and sheds new light on our understanding of the AIDS pathophysiology.

CD26 induces T-cell proliferation by tyrosine protein phosphorylation.

CD26 antigen distribution among lymphoid cells and its participation in the process of lymphocyte activation and proliferation has been widely documented. However, the molecular and biochemical mechanisms coupled to the CD26 molecule are not yet known. With different monoclonal antibodies (mAb) we have detected that approximately 56% of CD4+ and 35% of CD8+ cells from peripheral blood lymphocytes express CD26 and the expression of this antigen is required for antigen- but not for mitogen-induced proliferation unless exogenous interleukin-2 (IL-2) is added to the culture. The stimulation of nylon wool-separated T cells and T-cell clones by the anti-CD26 mAb, 134-2C2, induced tyrosine phosphorylation on a subset of proteins of 50,000, 46,000, 26,000, 24,000 and 21,000 MW. This pattern of phosphorylation was not affected by the presence of 12-myristate 13-acetate (PMA), although this cofactor is required for CD26-mediated IL-2 mRNA expression and T-cell proliferation. When a specific tyrosine kinase inhibitor, Tyrphostin, was used in CD4+ cells cultures stimulated with 134-2C2 and PMA, the proliferation and the expression of IL-2 mRNA were inhibited. Thus, protein tyrosine phosphorylation seems to play a major role in CD26-mediated T-cell proliferation.