The influence of Ouabain on human dendritic cells maturation.

Although known as a Na,K-ATPase inhibitor, several other cellular and systemic actions have been ascribed to the steroid Ouabain (Oua). Particularly in the immune system, our group showed that Ouabain acts on decreasing lymphocyte proliferation, synergizing with glucocorticoids in spontaneous thymocyte apoptosis, and also lessening CD14 expression and blocking CD16 upregulation on human monocytes. However, Ouabain effects on dendritic cells (DCs) were not explored so far. Considering the peculiar plasticity and the importance of DCs in immune responses, the aim of our study was to investigate DC maturation under Ouabain influence. To generate immature DCs, human monocytes were cultured with IL-4 and GM-CSF (5 days). To investigate Ouabain role on DC activation, DCs were stimulated with TNF-α for 48 h in the presence or absence of Ouabain. TNF-induced CD83 expression and IL-12 production were abolished in DCs incubated with 100 nM Ouabain, though DC functional capacity concerning lymphocyte activation remained unaltered. Nevertheless, TNF-α-induced antigen capture downregulation, another maturation marker, occurred even in the presence of Ouabain. Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained. Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.

mCD14 expression in human monocytes is downregulated by ouabain via transactivation of epithelial growth factor receptor and activation of p38 mitogen-activated protein kinase.

BACKGROUND AND AIMS: The steroid ouabain is found in plasma and in many mammalian tissues, and is now considered as a hormone. In the immune system, ouabain regulates a number of lymphocyte functions, but little is known about its effects on monocyte function. Monocytes are important for adequate immune responses. The aim of this work was to analyze the effect of ouabain on mCD14 expression, a surface molecule involved in the response against Gram-negative bacteria and phagocytosis. METHODS: Human peripheral blood mononuclear cells obtained from healthy donors were separated by density gradient centrifugation. Monocytes were separated by adherence and treated for 24 h with 100 nM ouabain. mCD14, CD1a and P-p38 expression was analyzed by flow cytometry. Inhibitors of cell-signaling pathways, i.e. SB202190, reduced glutathione, rottlerin, tyrphostin A23, genistein, chelerythrine chloride, PD98059, PP1 and Ly 294002, were used concomitantly with ouabain to observe their effect on mCD14 expression. RESULTS: Ouabain induced a significant decrease in mCD14 expression. This feature was not related to receptor endocytosis or cell death. Furthermore, mCD14 downregulation did not reflect a shift in differentiation into dendritic cells because this hormone failed to induce CD1a expression. Amongst several inhibitors of cell-signaling pathways triggered by ouabain, only epidermal growth factor receptor (EGFR) and p38 mitogen-activated protein kinase (MAPK) inhibitors (tyrphostin A23 and SB202109) significantly reverted the effect of ouabain on mCD14 expression. Accordingly, the levels of P-p38 were increased on monocytes after ouabain treatment. However, incubation with epidermal growth factor did not alter mCD14 expression. CONCLUSION: These findings suggest that ouabain downregulates mCD14 expression on monocytes through EGFR transactivation and p38 MAPK activation.

Profile of serum IL-1beta and IL-10 shortly after ovariectomy and estradiol replacement in rats.

Ovariectomy leads to progressive and significant increases in body weight gain and osteoporosis and is related to changes in serum and tissue cytokine profiles, such as observed in other models of overweight. We aimed to evaluate serum interleukin-1beta and interleukin-10 shortly after ovariectomy, before the establishment of overweight in rats. Female Wistar rats were submitted to ovariectomy, ovariectomy and estradiol replacement, or sham operation and compared with intact controls. Rats were killed 3, 6, 9, or 13 d after ovariectomy. Body mass and retroperitoneal fats were significant higher only 13 d after ovariectomy, and estradiol replacement to ovariectomized rats impaired both body mass and retroperitoneal fat gain. Shortly after ovariectomy (at 3 d) serum interleukin-1beta levels significantly increased in ovariectomized rats, treated or not with estradiol, while serum interleukin-10 levels increased only 9 d after ovariectomy. Our results suggest the existence of an important interplay between the immune system and ovarian function. This interplay occurs regardless of significant changes in adipose tissue compartment, as ovarian excision leads to short-term changes in the pattern of interleukin-1beta and interleukin-10 cytokine production that precede body weight gain and are not reverted by estradiol replacement.

Negative affect as a predisposing factor for cortisol release after an acute stress--the impact of unpleasant priming.

Glucocorticoids have a key role in stress responses. There are, however, substantial differences in cortisol reactivity among individuals. We investigated if affective trait and mood induction influence the reactivity to psychological stress in a group of 63 young adults, male (n=27) and female (n=36), aged ca. 21 years. On the experimental day the participants viewed either a block of pleasant or unpleasant pictures for 5 min to induce positive or negative mood, respectively. Then, they had 5 min to prepare a speech to be delivered in front of a video-camera. Saliva samples were collected to measure cortisol, and questionnaire-based affective scales were used to estimate emotional states and traits. Compared to basal levels, a cortisol response to the acute speech stressor was only seen for those who had first viewed unpleasant pictures and scored above the average on the negative affect scale. There were no sex differences. In conclusion, high negative affect associated with exposure to an unpleasant context increased sensitivity to an acute stressor, and was critical to stimulation of cortisol release by the speech stressor.

Independent regulation of ABCB1 and ABCC activities in thymocytes and bone marrow mononuclear cells during aging.

Aging modifies a number of functional and phenotypic parameters of cells from the immune system. In this study, the activities of two members of the superfamily of ATP-binding cassette (ABC) transport proteins, ABCB1 and ABCC (measured by rhodamine 123 efflux and Fluo-3 efflux respectively), were compared in murine bone marrow cells and thymocytes of young (3-4 weeks old), adult (2-3 months old) and old (18 months old) mice. ABCB1 activity was shown to be age regulated in murine bone marrow mononuclear cells and thymocytes. In the bone marrow, the increased amount of cells with ABCB1 activity observed in old mice was restricted to the c-kit(-)Sca-1(+) and c-kit(+)Sca-1(+) subpopulations. Only a small percentage of c-kit(+) cells in the thymus had ABCB1 activity, and this subpopulation increased with age. In the thymus, old age augmented this activity in the CD4(-) CD8(-) double-negative cells and in the CD4(+) and CD8(+) single-positive populations. The activity of another ABC transporter, the ABCC-related activity, was also modified by age in the bone marrow. However, the age-related increase was observed in the subpopulations were ABCB1 was not modified, namely the non-progenitor population (c-kit(-)Sca-1(-)cells) and c-kit(+)Sca-1(-) cells. Nearly, all thymocytes expressed the ABCC1 molecule in an active form and aging did not affect this pattern. This study demonstrates an independent upregulation of ABCB1 and ABCC activities during the aging process. The increases were observed in different subsets of cells but followed a developmentally regulated pattern. The functions played by these transporters and alterations in aging are discussed.

Bax expression and apoptotic cell death in Fanconi anaemia peripheral blood lymphocytes.

OBJECTIVE: Deregulated apoptosis might be involved in some of the features of Fanconi anaemia (FA). The possibility that the pro-apoptotic Bax protein could be involved in an increased susceptibility to apoptosis in FA patients was investigated. MATERIALS AND METHODS: Intracellular Bax expression, Bcl-2 expression (an anti-apoptotic protein) and cell death were analysed in 26 FA peripheral blood lymphocyte samples. RESULTS: Most FA samples (69%) displayed increased levels of Bax and were more susceptible to both spontaneous apoptosis and mitogen activation-induced cell death. Two subgroups were identified: one presented elevated levels of Bax (n = 18), whereas the other (n = 8), had Bax levels lower than controls. Two subgroups based on Bcl-2 expression were also identified: one with normal and another with high Bcl-2 expression. No inverse correlation was found between Bcl-2 levels and Bax expression. A clear difference in susceptibility to induced cell death could be observed between control and FA samples. The best correlation was observed between high levels of Bax and mitogen-induced apoptosis of cells; these displayed characteristics of necrosis secondary to apoptosis, suggesting that the intrinsic apoptotic pathway was being activated. CONCLUSION: Despite increased susceptibility to cell death induction, there was no correlation between Bax levels, chromosome breakage, haematological parameters or androgen therapy. The importance of apoptosis and Bax expression in the clinical development of FA awaits clarification.

Pomolic acid-induced apoptosis in cells from patients with chronic myeloid leukemia exhibiting different drug resistance profile.

Pomolic acid (PA) is a pentacyclic triterpene which has been previously described as active in inhibiting the growth of K562 cell line-originated from chronic myeloid leukemia (CML) in blast crisis-and its vincristine-resistant derivative K562-Lucena1. In this work, cells from CML patients were treated with PA and the apoptotic index was compared with the multidrug resistance (MDR) profile and clinical status of the patients. Our findings show that PA 12.5 microg/ml at 24 h (p = 0.000), at 48 h (p = 0.012) and at 72 h (p = 0.005) has a potent apoptotic index in CML cells as compared to mononuclear cells from healthy donors. PA was capable to induce apoptosis in cells from CML patients exhibiting functional MDR phenotype but not in P-glycoprotein expression. In addition, PA was effective in chronic as well as in blast phase of CML. Moreover, similar apoptotic index induced by PA was observed in low, intermediate and high-risk Sokal score as well as in samples from the group of patients with clinical resistance to interferon and/or imatinib and non-treated patients. These results suggest that PA may be an effective agent for the treatment of CML.

On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells.

BACKGROUND: That there is a correlation between cancer and procoagulant states is well-known. C6 glioma cell line was originally induced in random-bred Wistar-Furth rats and is morphologically similar to glioblastoma multiforme, the most common aggressive glioma resistant to therapeutic interventions. OBJECTIVES: In this study we analyzed the molecular mechanisms responsible for the highly procoagulant properties of C6 glioma cells. METHODS: The presence of tissue factor (TF) and phosphatidylserine (PS) in C6 cells was investigated by flow-cytometric and functional analyses. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. RESULTS: TF was identified by flow-cytometric and functional [factor (F) Xa formation in the presence of cells and FVIIa] assays. Alternatively, conversion of FX into FXa was also observed in the presence of C6 cells, FIXa and FVIIIa. This effect was both cell- and FVIIIa-dependent, being consistent with formation of the intrinsic tenase complex. C6 cells were also able to activate prothrombin in the presence of FXa and FVa, thus supporting formation of the prothrombinase complex. This ability was similar to positive controls performed with PS-containing vesicles. Accordingly, exposure of PS on C6 cells was demonstrated by flow cytometry employing specific anti-PS antibodies. In addition, annexin V, which blocks PS binding sites, inhibited FX and prothrombin conversion by their respective C6-assembled activating complexes. CONCLUSION: C6 glioma cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant TF exposure and from the presence of the anionic lipid PS at the outer leaflet of cell membrane. Therefore, this animal cell line may be used to explore new aspects concerning the role of blood coagulation proteins in tumor biology, especially those affecting the central nervous system.

Methylene blue is more toxic to erythroleukemic cells than to normal peripheral blood mononuclear cells: a possible use in chemotherapy.

Methylene blue (MB) is a phenothiazine with radio and photosensitizing properties and anti-tumoral activity. Our group has shown that MB was capable of inhibiting the in vitro growth of erythroleukemic cells with multidrug resistance (MDR). However, there are no studies comparing the cytotoxicity of this molecule for normal and tumoral cells. In this work, the cytotoxicity of MB was measured by MTT method in erythroleukemic and melanoma lineages, comparing it with that of normal cells:lymphocytes and melanocytes. MB was more cytotoxic for tumoral cells; however, there was no difference between erytroleukemic cells with or without MDR phenotype. Lymphocytes and erythroleukemic cells were much more sensitive to the effects of MB than melanoma cells and melanocytes. The proliferation of phytohemagglutinin-activated lymphocytes was inhibited when 3H-thymidine incorporation to DNA was measured. We tried to analyze whether the cells were dying, via apoptosis or necrosis, using Anexin-V and propidium iodide. Despite higher levels of Anexin-V, it was not possible to distinguish necrosis from apoptosis, as the fluorescence of MB is in the same channel as propidium iodide. The production of hydrogen peroxide was measured by cytometry using dihydrorhodamine 123 (DHR). Despite the erythroleukemic cells and lymphocytes being capable of producing free radicals, there was no relation between the production and the sensitivity of various cells to MB. Our results suggest that MB should be used as a chemotherapeutic agent, because of its preferential cytotoxic effects over tumor cells, considering the fact that MDR cells are also sensitive, and due to its radio and photosensitizing activities.

Oral mucosa alterations induced by cyclosporin in mice: morphological features.

BACKGROUND AND OBJECTIVE: The mechanisms involved in the pathogenesis of cyclosporin A-induced gingival hyperplasia are not well understood. The present work aimed at developing a mouse model with the characteristics of the human process, i.e. time of appearance, dose dependency and the capacity of developing in a variety of genetic backgrounds. This model would present the advantages of using a very well known animal species, small and easy to handle, with a number of experimental reagents (antibodies, etc.) already available against its products. METHODS: Three different strains of mice were used: CBA, F1(C57Bl x DBA), Balb/c. Groups of mice received different concentrations of cyclosporin A (CSA) (10 mg/kg, 25 mg/kg and 40 mg/kg body weight) intraperitoneally five times a week. Anatomical and histological alterations were recorded at various time intervals. RESULTS: All strains of mice presented gingival hyperplasia after 8 weeks of CSA treatment. A dose-dependency was observed with regard to the time of first appearance of alterations. Increased redness was seen in all animals at the sixth week, independent of the dosage used. Histologic examination exhibited increased vascularization, epithelial and connective tissue thickening, edema and a mononuclear infiltrate. CONCLUSIONS: It was possible to develop CSA-induced gingival hyperplasia in mice with the characteristics described in humans and other species. The use of this animal model may help in the elucidation of the process involved in CSA-induced gingival overgrowth.

p53 gene analysis in childhood B non-Hodgkin's lymphoma.

CONTEXT: Mutations or deletions in the tumor-suppressor gene p53 are among the commonest genetic changes found in human neoplasms including breast, lung and bowel cancers. In hematological malignancies, p53 is most often mutated in Burkitt's lymphoma, with p53 mutations present in 30 to 40% of tumor samples and in 70% of cell lines. OBJECTIVE: To analyze the p53 gene alterations in child patients with B non-Hodgkin's lymphoma. DESIGN: Descriptive study. SETTING: Tertiary oncology care center. PARTICIPANTS: The study investigated 12 patients with childhood B non-Hodgkin's lymphoma (Burkitt's lymphoma). Screening for p53 mutations was done by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of exon 5 to 8/9 of the gene. RESULTS: Abnormal polymerase chain reaction-single strand conformational polymorphism migration pattern was observed in 4 patients (33.3%), one on exon 6 and three on exon 7. Positive cases included 2 patients who died from disease. CONCLUSION: These preliminary results suggest that p53 mutations are quite frequent in children with Burkitt's lymphoma and may play a role in lymphoma genesis or disease progression.

Reduced glutathione protect cells from ouabain toxicity.

It is widely accepted that a prolonged ouabain blockade of the Na(+),K(+)-ATPase makes cells detach from each other and from the substrate, leading to their death and that cellular resistance to ouabain is due to the presence of isoforms of Na(+),K(+)-ATPase with low affinity to this glycoside. In the present work the effect of reduced glutathione in the response of two types of renal cells to ouabain: MDCK, a ouabain-sensitive cell line and Ma104, a ouabain-resistant one, was studied. Glutathione protected MDCK cells from ouabain toxicity and inhibition of glutathione synthesis by L-buthionine-S,R-sulfoximine sensitized Ma104 cells to ouabain. As glutathione is involved with multidrug resistance (MDR) in cells expressing the multidrug resistance-related protein MRP1 and as Ma104 cells have a MDR phenotype, it was investigated whether Ma104 cells express this protein. The expression of the MRP1-mRNA in Ma104 cells was detected by reverse transcriptase-polymerase chain reaction and ribonuclease protection assay, and the protein was detected by Western blotting and immunofluorescence. Treatment of Ma104 cells with ouabain increased MRP1-mRNA expression and altered the localization of MRP1 in these cells. Our results suggest that some cells may have mechanisms to protect themselves from ouabain toxicity and that MRP1 may have a role in controlling the toxic effects of ouabain.

Multidrug resistance in tumour cells: characterization of the multidrug resistant cell line K562-Lucena 1.

Multidrug resistance to chemotherapy is a major obstacle in the treatment of cancer patients. The best characterised mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein. However, the resistance process is multifactorial. Studies of multidrug resistance mechanisms have relied on the analysis of cancer cell lines that have been selected and present cross-reactivity to a broad range of anticancer agents. This work characterises a multidrug resistant cell line, originally selected for resistance to the Vinca alkaloid vincristine and derived from the human erythroleukaemia cell K562. This cell line, named Lucena 1, overexpresses P-glycoprotein and have its resistance reversed by the chemosensitisers verapamil, trifluoperazine and cyclosporins A, D and G. Furthermore, we demonstrated that methylene blue was capable of partially reversing the resistance in this cell line. On the contrary, the use of 5-fluorouracil increased the resistance of Lucena 1. In addition to chemotherapics, Lucena 1 cells were resistant to ultraviolet A radiation and hydrogen peroxide and failed to mobilise intracellular calcium when thapsigargin was used. Changes in the cytoskeleton of this cell line were also observed.

Methylene blue reverts multidrug resistance: sensitivity of multidrug resistant cells to this dye and its photodynamic action.

Photodynamic action has been advocated as an alternative treatment of tumors but the most common used dyes, hematoporphyrin derivatives, are substrate for P-glycoprotein. This study investigated the MDR-reverting properties of methylene blue (MB) and compared the sensitivity to its photodynamic action (PDA) in five cell lines that either express or do not express the MDR phenotype. MB was able to revert the MDR phenotype and there was no difference in sensitivity to MB-PDA between MDR and non-MDR cells, suggesting that MB has the advantage of being used simultaneously as a MDR reverser and a photodynamic agent.

Differences in sensitivity to UVC, UVB and UVA radiation of a multidrug-resistant cell line overexpressing P-glycoprotein.

Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells, selected for resistance to one chemotherapeutic agent, simultaneously acquire resistance to several apparently unrelated drugs. The MDR phenotype is multifactorial. The best-studied mechanism involves the expression of a membrane protein that acts as an energy-dependent efflux pump, known as P-glycoprotein (Pgp), capable of extruding toxic materials from the cell. In this work, resistance to UVA radiation, but not to UVC nor UVB, was observed in an MDR leukemia cell line. This cell line overexpresses Pgp. To study the role of Pgp in the resistance to UVA radiation, two MDR modulators or reversing agents (verapamil and cyclosporin A) capable of blocking Pgp activity were used. Cell viability was assessed and the techniques of flow cytometry and fluorescence microscopy were employed to measure the extrusion of rhodamine 123 by the efflux pump. The results show that MDR modulators did not modify the resistance to UVA radiation. Furthermore, although cell viability was not significantly altered, Pgp function was impaired after UVA treatment, suggesting that this glycoprotein may be a physical target for oxidative damage, and that other factors may be responsible for the UVA resistance. In agreement with this, it was found that the resistant cell line presented a higher catalase activity than the parental (non-MDR) cell line.

The in vivo effect of the administration of resistance-modulating agents on rhodamine 123 distribution in mice thymus and lymph nodes.

P-glycoprotein (Pgp) has been widely associated with the multidrug resistance phenotype. Nevertheless, this protein has been detected in many normal tissues and cells, including liver, kidney, endothelial cells that constitute the hematological barrier of the brain and testes, and cells from the immune system. Many in vitro models have been used to study drugs that modulate Pgp activity and the multidrug resistance phenomenon. In the present work, we investigate the in vivo effects of resistance-modulating agents on lymphoid organs. Rhodamine 123 (Rho123), a well-known Pgp substrate, was administered to mice, and the fluorescence level in thymus and lymph node cells measured. The fluorescence level on these organs showed a dose-dependent response. Cyclosporin A (CSA), Verapamil (VP) and Trifluoperazine (TFP), three resistance-modulating agents, were administered to mice 1 h prior to 1 mg/kg Rho123 administration. Surprisingly, VP (10 mg/kg) and TFP (750 microg/kg) did not modulate Rho123 retention by thymus and lymph node cells. CSA (50 mg/kg) was the only drug that increased the fluorescence level in both organs. These results point out to the need of a wider study on the in vivo effects of resistance-modulating agents in different organs and systems.

Cyclosporin A and trifluoperazine, two resistance-modulating agents, increase ivermectin neurotoxicity in mice.

The P-glycoprotein expressed in the blood-brain barrier has been associated with the restricted access of many compounds to the central nervous system. Mice lacking the mdr1a P-glycoprotein gene show an accumulation of various drugs in brain tissues. P-glycoprotein is also correlated with the phenomenon of multidrug resistance in tumour cells. To investigate the effects of drugs that modulate multidrug resistance in the selective permeability of the blood-brain barrier, mice were treated with cyclosporin A or trifluoperazine plus ivermectin, a P-glycoprotein substrate, that has a limited access to the central nervous system. When mice received an injection of cyclosporin A (50 mg/kg, intraperitoneally) or trifluoperazine (750 microg/kg, intraperitoneally) one hour prior to the administration of ivermectin (10-15 mg/kg, intraperitoneally) there was an increase in the acute toxicity of ivermectin. HPLC analysis of brain tissues indicated that the ivermectin brain concentration was 2.5 times higher when mice were previously treated with cyclosporin A (50 mg/kg). These results suggest that attention should be given to the side effects of drugs that interact with P-glycoprotein and are commonly used clinically and also to the possibility of creating a pharmacological gap in the blood-brain barrier that allows the access of chemotherapeutic drugs to brain tumours.

Expression of functionally P-glycoprotein in MA104 kidney cells.

Rhesus monkey kidney MA104 cells are a polarized epithelium with some unusual characteristics, including a resistance to ouabain, although their Na(+)-K(+)-ATPase has normal affinity with this drug. This work suggests that MA104 cells have high expression of functionally P-glycoprotein in their membranes. This was established using four complementary methods to investigate the expression and function of P-glycoprotein in these cells. MA104 cells were strongly resistant to vincristine, which could be reversed by three known P-glycoprotein modulators: verapamil, cyclosporin A and trifluoperazine. In addition, MA104 cells accumulate little rhodamine 123, and the incubation with verapamil increased this accumulation. The mdr1-mRNA was detected by reverse transcription-polymerase chain reaction and a subcloned 283-bp product was identified. Its nucleotide sequence was compared with the related region of human mdr1, showing a high identity (96%) between the two sequences. The expression of P-glycoprotein in the cell membrane was observed by Western blot and immunofluorescence. The results taken together suggest that MA104 cells intrinsically have a high expression of functionally P-glycoprotein in their membranes.

Natural killer cell activity in Hodgkin's disease patients undergoing radiation therapy or chemotherapy and radiation therapy.

Natural killer (NK) cell activity in peripheral blood mononuclear cells (PBMCs) from patients with Hodgkin's disease was studied using 4 h 51Cr release assay and K562 cells as sensitive targets. PBMCs were obtained from 15 previously untreated patients at different stages of their disease. PBMCs were also obtained from 46 patients treated by radiation therapy or combined chemotherapy and radiation therapy. Twenty healthy age-matched volunteer donors were used as controls to the treated patients. For these normal donors the mean cytotoxicity was 24.8 +/- 5.67% at a 100:1 effector-target cell ratio; and 43.7 +/- 12.1% for the treated cancer patients. Fifteen healthy age-matched volunteer donors were used as controls to the untreated patients. The mean cytotoxicity for these normal donors was 20.8 +/- 3.61% at a 100:1 effector-target cell ratio; and 37.6 +/- 6.65% for the previously untreated cancer patients. The mean cytotoxicity for all 35 normal donors was 23.1 +/- 5.22% at a 100:1 effector-target cell ratio. Most treated patients (93.5%) had a complete response to therapy and a significant difference was found between the mean cytotoxicity of the whole group (46 treated patients), compared with controls (P < 0.001). A significant difference (P < 0.05) was also observed when the same 11 patients were studied before and after treatment.

Ouabain induces apoptosis on PHA-activated lymphocytes.

Apoptotic cell death plays a critical role in immune system homeostasis, and c-myc protooncogene deregulated expression is a component of this programmed genomic response. Pharmacological intervention and modulation of peripheral lymphocytes apoptosis would have important implications. The present results indicate that ouabain, a specific inhibitor of Na+K(+)-ATPase, promotes an increased expression of c-myc mRNA, and induces apoptosis in PHA-stimulated lymphocytes. Furthermore, this ouabain-induced apoptosis cannot be counteracted by the addition of exogenous IL-2.