Oxidative Upregulation of Bcl-2 in Healthy Lymphocytes SILVIA CRISTOFANON,a,b SILVIA NUCCITELLI,b MARIA D’ALESSIO,b FLAVIA RADOGNA,b MILENA DE NICOLA,b ANTONIO BERGAMASCHI,c CLAUDIA CERELLA,b ANDREA MAGRINI,c MARC DIEDERICH,a AND LINA GHIBELLIb a Laboratoire de Biologie Moléculaire et Cellulaire du Cancer (LBMCC), Fondation Recherche sur le Cancer et les Maladies du Sang, Hôpital Kirchberg, L-2540 Luxembourg, Luxembourg b Dipartimento di biologia, Universitá di Roma “Tor Vergata,” 00133 Roma, Italy c Cattedra di Medicina del Lavoro, Universitá di Roma “Tor Vergata,” 00133 Roma, Italy ABSTRACT: In many cell systems, pharmacological glutathione (GSH) depletion with the GSH neosynthesis inhibitor buthionine sulfoximine (BSO) leads to cell death and highly sensitizes tumor cells to apoptosis induced by standard chemotherapeutic agents. However, some tumor cells upregulate Bcl-2 in response to BSO, thus surviving the treatment and failing to be chemosensitized. Cell lines of monocytic and lymphocytic origins respond to BSO treatment in an opposite way, lymphocytes being chemosensitized and unable to transactivate Bcl-2. In this article we investigate the response to BSO of lymphocytes freshly isolated from peripheral blood of healthy donors. After ensuring that standard separation procedures do not alter per se lymphocytes redox equilibrium nor Bcl-2 levels in the first 24 h of culture, we show that BSO treatment promotes the upregulation of Bcl-2, with a mechanism involving the increased radical production consequent to GSH depletion. Thus, BSO treatment may increase the differential cytocidal effect of cytotoxic drugs in tumor versus normal lymphocytes. KEYWORDS: Bcl-2; buthionine sulfoximine; apoptosis; glutathione; lymphocyte Address for correspondence: Marc Diederich, Fondation Recherche sur le Cancer et les Maladies du Sang, Laboratoire de Biologie Moleculaire, et Cellulaire du Cancer (LBMCC), Hopital Kirchberg, 9, rue Edward Steichen, L-2540 Luxembourg. Voice: +352-2468-4040; fax: +352-24684060. e-mail: marc.diederich@lbmcc.lu C 2006 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1091: 1–9 (2006). doi: 10.1196/annals.1378.049 1 2 ANNALS NEW YORK ACADEMY OF SCIENCES INTRODUCTION Among the biochemical mechanisms responsible for apoptotic cell death of human tumor cell, particular attention has been devoted to those related to oxidative stress generated by oxygen-free radicals and peroxides.1,2 The main features of oxidative stress are the loss of protective action of biological scavengers, such as superoxide dismutase and glutathione (GSH). This results in an increased level of peroxides, hydroperoxides, and free radical. The importance of intracellular GSH in the pathology of disease, particularly cancer, has long been appreciated.3 However, the ubiquitous nature of GSH has made it difficult to ascribe to a specific molecular mechanism in disease accomplishment. In many cell systems, pharmacological GSH depletion with the GSH neosynthesis inhibitor buthionine sulfoximine (BSO) leads to cell death and highly sensitizes tumor cells to apoptosis induced by standard chemotherapeutic agents.4 For these reasons, BSO is widely used in clinical practice as a chemosensitizing agent.5 We had shown that unlike most tumors, two human tumor cell lines (U937 monocyte, and Hep G2, hepatocyte) survive to BSO, not because BSO is unable to elicit an apoptotic response in these cells, but because the apoptotic process is stopped after cytochrome c release and before caspase activation, due to the development of an adaptive response.6 We studied the mechanisms of such an adaptation and found that as a response to BSO, U937 upregulates Bcl-2 mRNA and protein levels by a mechanism possibly involving NF-B transcription factor.7 Moreover, BSO-dependent Bcl-2 upregulation is associated with the ability to survive the BSO; interestingly, GSH depletion upregulates Bcl-2 in BSO-resistant but not in BSO-sensitive cells.7 A fundamental problem in the field of lymphoid neoplasia concerns the relationship between normal immune elements in patients with lymphomas and lymphocytic leukemias, and the tumor cells, which themselves constitute a part of the host’s immune system.8 Bl-41, a cell line of lymphocytic origin deriving from an Epstein–Barr-negative Burkitt lymphoma, does not develop an adaptive response to BSO treatment, being (a) unable to transactivate Bcl-2, (b) induced to apoptosis, and (c) chemosensitized by BSO.7 This suggests that BSO might act as a chemosensitizer and improve the treatment of some lymphomas. Here, we investigate the response to BSO of lymphocytes freshly isolated from peripheral blood of healthy donors, with the goal of understanding whether the use of BSO as an adjuvant may help to increase the differential cytocidal effect of antitumor therapy between normal and tumor counterparts. MATERIALS AND METHODS Mononuclear White Blood Cells Isolation Peripheral blood mononucleated cells (PBMC) were isolated from the anticoagulated peripheral blood of 22 healthy adult human donors (13 men CRISTOFANON et al.: OXIDATIVE UPREGULATION OF BCL-2 3 and 9 women) using the standard Ficoll–Hypaque (Sigma) density separation method. After isolation, PBMC were washed, counted, resuspended at cell density of 1 × 106 cells/mL in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/mL penicillin, and streptomycin and kept in a controlled atmosphere (5% CO 2 ) incubator at 37◦ C. All the experiments were performed on mixed mononuclear cells, that is, lymphocytes+monocytes, distinguished by differential labeling upon flow cytometric analysis (see below). Surface Staining The expression of CD3-CD19 on lymphocyte was analyzed by adding 5 L phycoerythrin (PE)-conjugated anti-CD3, 5 L PE-conjugated anti-CD19 (Becton Dickinson) to leukocyte pellets. The suspensions were incubated in ice for 20 min in the dark and then washed twice in phosphate-buffered saline (PBS)-EDTA. The cells were finally resuspended in 0.5 mL of PBS and processed in a DAKO Galaxy flow cytometer. Statistics were elaborated in 50,000 events/sample by FlowMax software. Flow Cytometric Analysis FlowMax software permitted to study on the same dot plot FSC versus SSC quantization and counting of singular populations of monocytes and lymphocytes. Gating the area of labeled lymphocytes allowed to get information on parameters (abundance and fluorescence, corresponding to GSH, ROS, and Bcl-2 levels) of the specific area, as shown in FIGURE 1 A. Cell Treatments GSH depletion was performed by inhibiting GSH neosynthesis with 1 mM BSO (Sigma), which depletes PBMC in 18–24 h. Vitamin E analog Trolox C (Fluka) was used as a radical scavenger9 ; it was added to the cell culture at the final concentration of 500 M (30 min prior to BSO when used together). Bcl-2 Determination Cells were fixed, permeabilized, and stained with anti-Bcl-2 monoclonal antibody (Calbiochem Novabiochem Corp., San Diego, CA) according to the manufacturer’s instruction.7 Detection was done with FITC-conjugated antibodies and processed in a DAKO Galaxy flow cytometer. Statistics were elaborated in 50,000 events/sample by FlowMax software. Mean values given 4 ANNALS NEW YORK ACADEMY OF SCIENCES FIGURE 1. Identification of lymphocytes and quantification of basal parameters. (A) The area corresponding to CDX+ cells (B+T lymphocytes) is indicated; the population maintains a homogenous and compact distribution throughout the experimental time. (B) GSH, ROS, and Bcl-2 levels, measured as described in the section on “Materials and Methods,” are shown at 0, 12, and 18 h of culture. The separation protocol does not change any oxidative characteristics in explanted lymphocytes. The weak changes are not statistically significant. All values are expressed as fold increase with respect to control cell sample at 0 h of culture, which was considered = 1. Results are the mean of 18 experiments performed on blood samples of different individuals ± SD. by this analysis were used for further elaboration. For comparison between different experiments, the value of each treated cell sample was compared with the value of the control cell sample (untreated cells after separation protocol), which was considered equal to 1. The values were then given as a fold increase with the respect control. CRISTOFANON et al.: OXIDATIVE UPREGULATION OF BCL-2 5 GSH Determination GSH intracellular levels were detected with Orto-ftaldeide (OPTA, Molecular Probes)10 (ec. = 340 nm-em. = 445 nm). OPTA was added to the cell suspension at the final concentration of 50 M. The suspensions were incubated at 37◦ C for 20 min in the dark and then washed. The cells were finally resuspended in PBS and processed in a DAKO Galaxy flow cytometer. Statistics were elaborated in 50,000 events/sample by FlowMax software. Mean values from this analysis were used for further elaboration. For comparison between different experiments, the value of each treated cell sample was compared with the value of the control cell sample (untreated cells after separation protocol), as fold increase (control = 1). Determination of ROS Production ROS levels were detected with 2-7-dichloro-fluorescein-diacetate (DHCFDA) (ec. = 490 nm-em. = 520 nm), which was added to the cells at the final concentration of 10 M. DHCFDA fluoresces only when oxidized. The suspensions were incubated at 37◦ C in the dark for 20 min and then washed. Cells were finally resuspended in PBS and processed in a DAKO Galaxy flow cytometer. Statistics were elaborated in 50,000 events/sample by FlowMax software. Mean values given by this analysis were used for further elaboration. For comparison between different experiments, the value of each treated cell sample was compared with the value of the control cell sample (untreated cells after separation protocol), as fold increase (control = 1). Statistical Analyses Statistical analyses were performed using Student’s t-test for unpaired data, and P values less than 0.05 were considered significant. Data are presented as fold increase versus 0 h of treatment cells ± SD. RESULTS Separation Procedures Do Not Stimulate a Significant Alteration of Oxidative Parameters and Bcl-2 Levels in Explanted Lymphocytes The procedures required to purify the different types of white blood cells present in the regular buffy coats are quite harsh and may lead to cell alterations as it occurs, that is, for redox parameters in monocytes (Cristofanon et al., in preparation). To determine whether separation protocol and culture 6 ANNALS NEW YORK ACADEMY OF SCIENCES conditions could alter the redox state of lymphocytes, we analyzed the levels of GSH and radicals, with OPTA and DHCFDA, respectively, in the lymphocytes population for the first 24 h of culture. No significant alterations in GSH levels were detectable in 24 h; intracellular-free radicals levels appeared slightly but not significantly increased at 18 h of culture (FIG. 1 B). Since we have shown that Bcl-2 protein level may respond to redox alterations, we expect that in these quite stable conditions Bcl-2 levels remained unaltered. FIGURE 1 B shows that indeed Bcl-2 levels are stable in cultured lymphocytes. BSO Induces Bcl-2 Upregulation in Lymphocytes as a Response to ROS Production In order to explore the response of lymphocytes to redox disequilibrium, purified lymphocytes were treated with BSO. FIGURE 2 A shows that GSH is depleted by about 35% after 18 h of BSO in the treated lymphocytes. At the same time points, BSO treatment strongly stimulated ROS production (FIG. 2 A). Thus, depleting the GSH we created an oxidative condition inside the cells; in these oxidative conditions, BSO induces a sudden Bcl-2 upregulation, as shown in FIGURE 2 A. To investigate whether the strong BSO-dependent Bcl-2 upregulation was due to ROS production or to the decreased availability of GSH, we scavenged the ROS produced by BSO with Trolox C, which does not change the kinetic of depletion of GSH, but efficiently scavenges the BSO increased ROS levels (data not shown). FIGURE 2 B shows that radicals scavenging revert the BSOdependent upregulation of Bcl-2, showing that in this cell system it is a response to radical production. DISCUSSION The theoretical basis for the use of BSO as a chemosensitizer stems from the general findings that cells deprived of GSH are less prone to extrude xenobiotics (via GSH-S-transferase), thus bypassing the problem of multidrug-resistance, and to resist to apoptosis, due to reduced antioxidant ability and reduced levels of the antiapoptotic protein Bcl-2. Indeed, most cells respond to GSH depletion by downregulating Bcl-2: this is probably due to an increased breakdown of Bcl-2 protein in an oxidizing environment.11 The different susceptibility to BSO treatment in different cellular lines, is probably linked to the intrinsic ability of some cells to adapt to GSH depletion by activating specific survival pathways.7 We have shown that BL-41 (human B cell Burkitt lymphoma cell line), unlike U937(monocytic cell line), is induced to CRISTOFANON et al.: OXIDATIVE UPREGULATION OF BCL-2 7 FIGURE 2. BSO upregulates Bcl-2 in healthy lymphocytes as a response to ROS production. (A) GSH, ROS, and Bcl-2 levels ROS levels in lymphocytes treated with BSO 1 mM. The increase of ROS and Bcl-2 levels at 18 h of cells treated with BSO is statistically significant (P < 0.05) with respect to 0 and 12 h. All values are expressed as fold increase with respect to control cell sample at 0 h of culture, which was considered = 1. Results are the mean of 18 experiments performed on blood samples of different individuals ± SD. (B) Levels of Bcl-2 in lymphocytes untreated and treated for 18 h with BSO, BSO+TroloxC, and Trolox C. Bcl-2 increase in cells treated with BSO with respect to untreated; and Bcl-2 decrease in the cells treated with BSO+Trolox C with respect to BSO alone are statistically significant (P < 0.05). All values are expressed as fold increase with respect to control cell sample at 0 h of cultures, which was considered = 1. Results are the mean of 18 experiments performed on blood samples of different individuals ± SD. apoptosis by 1 mM BSO.7 In this study we were interested to understand if the inability of BL-41 to respond to GSH depletion by upregulating Bcl-2 is a histotypic idiosyncrasy. Modulation of Bcl-2 protein levels as an adaptive response should conceivably be an important regulatory mechanism for cell survival in 8 ANNALS NEW YORK ACADEMY OF SCIENCES conditions of sudden environmental changes. Bcl-2 upregulation has been shown to occur in physiologically regulated situations, such as the rescue of target cells by prosurvival cytokines (i.e., NGF).12,13 The upregulation of Bcl-2 as a response to damaging situations, such as stressing disulfide disturbances and oxidative stress, has been documented.14 Commonly used cells separation procedures do not induce activation and changes on oxidative profile (GSH extrusion and ROS production) of healthy lymphocytes, unlike monocytes (Cristofanon et al., in preparation); redox challenges are among the most important trigger of leukocyte activation; indeed, ROS production and the ability to live in an oxidative environment is mandatory for an appropriate inflammatory response of monocyte; less for lymphocytes. It is important to stress that in the experiments here described the two types of mononuclear leukocytes were not physically separated: thus, any radical present in the extracellular medium, even though produced by monocytes, was able to cross lymphocytes plasma membrane. Thus, the different behavior toward radical challenging might be explained by hypothesizing that perhaps lymphocytes need more specific triggers than oxidative stress to be activated. The lack of proradical effect of the separation procedures per se allowed us to study the response of BSO-induced oxidative stress on the Bcl-2 levels without problems of overlapping events. We found that BSO treatment induces the upregulation of Bcl-2 in healthy lymphocytes, unlike what occurs in their tumor counterpart, that is, BL-41. This means that either there is no such factor like an histotypic idiosyncrasy allowing to predict cell response to oxidative challenge, in terms of Bcl-2 upregulation; or, tumor transformation implies the loss of the ability to respond to an oxidative environment building up a defense that may allow longer survival time for the working lymphocyte in the inflammation site that conceivably is an oxidizing one. As a last point, we want to stress that the mechanism at the basis of Bcl-2 upregulation as a response to GSH depletion is still unsolved. In this study, we show that it is not the decreased GSH concentration, but rather the increase in free radicals, which acts as the stimulus for Bcl-2 upregulation. 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