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Cell Biology International (2006) 30, 727–732 (Printed in Great Britain)
CD38 expression enhances sensitivity of lymphoma T and B cell lines to biochemical and receptor-mediated apoptosis
Armando Gregorinia*, Marco Tomasettib, Cristina Cintib, Daniela Colombac and Stella Colombad
aIstituto di Psicologia “L. Meschieri”, Università di Urbino “Carlo Bo”, via O. Ubaldini 17, 61029 Urbino (PU), Italy
bDipartimento di Patologia Molecolare e Terapie Innovative, Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy
cIstituto di Clinica Medica I, Università di Palermo, P.zza delle Cliniche 2, 90127 Palermo, Italy
dIstituto di Ecologia e Biologia Ambientale, Università di Urbino “Carlo Bo”, Via I. Maggetti 22, 61029 Urbino (PU), Italy


Abstract

CD38 has been widely characterised both as an ectoenzyme and as a receptor. In the present paper, we investigated the role of CD38 as possible modulator of apoptosis. CD38-positive (CD38+) and negative (CD38) fractions, obtained by sorting CD38+ cells from lymphoma T (Jurkat) and lymphoma B (Raji) and by transfecting lymphoma LG14 and myeloid leukemia K562 cell lines, were used. Cellular subpopulations were exposed to different triggers (H2O2, UV-B, α-TOS and hrTRAIL) and the extent of apoptosis was determined by Annexin V-FITC/PI assay. Our data showed that, in lymphoma cells, propensity to apoptosis was significantly linked to CD38 expression and that, remarkably, such response was independent of the nature of the trigger used. Inhibition of CD38 expression by antisense oligonucleotides treatment resulted in CD38-silenced fractions which were as prone to apoptosis as CD38 ones. Notably, susceptibility of K562 to apoptosis-inducing challenges was not affected by CD38 expression.


Keywords: CD38, Apoptosis, Lymphoma T cells, Lymphoma B cells, Myeloid leukemia cells.

*Corresponding author. Tel.: +39 722 303438; fax: +39 722 303436.


1 Introduction

Human CD38 is a 42–45kDa single chain type II transmembrane glycoprotein encoded by a large, complex, single copy gene that extends over 60 kilobases (kb) and maps to 4p15. This surface molecule consists of a short amino-terminal cytoplasmic tail, a single membrane spanning region, and a long extracellular carboxy-terminal domain. CD38 is widely expressed at variable levels in tissues and cells, especially in leukocytes, mostly during early differentiation and activation (Mehta et al., 1996).

The biological role of the molecule is still controversial. However, some of its functions have progressively been characterised: CD38 acts as a multifunctional ectoenzyme (Howard et al., 1993; De Flora et al., 1998) and plays a role in cell adhesion, signal transduction and calcium signalling (Shubinsky and Schlesinger, 1997; Deaglio et al., 1999).

In human lymphocyte B, T and natural killer (NK) cells, the CD38 signalling pathway depends on its physical and functional interactions with specialised signalling molecules, such as the B-cell receptor (BCR) complex, the CD3 complex and CD16 (Deaglio et al., 2002). Other molecules that share significant structural and functional homology with CD38 have been identified in humans, mice and rats and the finding that their coding genes are synthenic and located in the same chromosome region suggests the existence of a new family of related proteins involved in the regulation of a cell's life and death (Ferrero and Malavasi, 1997). CD38 also takes part in activation, proliferation and apoptosis of both normal and leukemic blood cells (Malavasi et al., 1992), but to date there is only limited understanding of its role in the modulation of the apoptotic process.

Programmed cell death (PCD) or apoptosis is a physiological process leading to the elimination of useless and harmful cells, which is very important for preserving tissue homeostasis in multicellular organisms. Recent reports indicate that apoptosis is regulated by the complex interaction of several families of proteins that have been conserved throughout evolution. PCD causes characteristic morphological changes in the cells, which include the release of small vesicles derived from the cytoplasmic membrane (the phenomenon known as blebbing), shrinkage of the cell and detachment from the surrounding structures, chromatin condensation, and nuclear and cellular fragmentation. The process ends with the phagocytosis of dead cells and apoptotic bodies either by neighbouring cells or by specialised phagocytes (Green, 2000; Hengartner, 2000).

In this study, we investigated the role of human CD38 as a possible modulator of apoptosis induced by biochemical and immunological triggers in vitro. To this aim, lymphoma T (Jurkat), lymphoma B (Raji, LG14) and myeloid leukemic K562 cell lines were used. Both Jurkat and Raji cells were fractionated into two different subpopulations sorted by their CD38 expression (Jurkat CD38+ and CD38; Raji CD38+ and CD38). LG14 and K562 were transfected with a CD38 cDNA carrying vector to obtain LG14 CD38+ and K562 CD38+ cells. CD38+ and CD38 subpopulations from all cell lines were stimulated with H2O2, UV-B, α-TOS, hrTRAIL and the extent of apoptosis was determined by flow cytometric analysis (Annexin V-FITC/Propidium Iodide assay). Moreover, to establish whether apoptotic responses were dependent on CD38 expression, we re-evaluated susceptibility to apoptosis of CD38+ cells after silencing CD38 gene expression by antisense oligonucleotide treatment. Our findings provided additional evidence of a strong correlation between the apoptotic process and CD38 expression.

2 Materials and methods

2.1 Cell cultures

Human Burkitt lymphoma (Raji), T cell acute lymphoblastic lymphoma (Jurkat), B-lymphoblastoid (LG14) and myeloid erythroleukemic (K562) cell lines were used. Cells were cultured in RPMI 1640 medium with 10% heat inactivated fetal bovine serum, 2mM l-glutamine, 100U/ml penicillin and 100μg/ml streptomycin in an atmosphere of 5% CO2 in humidified air at 37°C. In all experiments, exponentially growing cells were utilized.

Jurkat and Raji cells were enriched for CD38+ cells using either limiting dilution or magnetic microbeads (Dynal, Oslo, Norway). Two subpopulations, CD38+ and CD38, were obtained for each cell line. CD38 expression was routinely analysed by indirect immunofluorescence (IIF) and flow cytometry using an anti-CD38 monoclonal antibody (mAb) followed by a secondary FITC-conjugate IgG (Caltag, Burlingame, CA, USA). Re-analysis of CD38+ sorted cells indicated >95% purity (Fig. 1A).


Fig. 1

Flow cytometric re-analysis of CD38+ and CD38 sorted cells. (A) Raji CD38 (dark profile) and CD38+ cells (white profile). (B) LG14 CD38 (dark profile) and CD38+ cells (white profile). Similar results were obtained with Jurkat and K562 CD38 and CD38+ subpopulations.


2.2 CD38 cloning and transfection

CD38 cDNA contained in a pCDM8 plasmid (from E. Ferrero, University of Torino, Italy) was amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers designed according to the published CD38 sequence (GeneBank Accession M34461):

forward primer: 5′-CTC TCT TGC TGC CTA GCC TC-3′

reverse primer: 5′-TCA GAT CTC AGA TGT GCA AGA TGA-3′

PCR amplification was carried out using an automated DNA thermal cycler (Perkin–Elmer, Monza, Italy) for 30 cycles following an initial denaturation of 5min at 94°C. The reaction product was visualized by electrophoresis on a 1.5% agarose gel containing tris–borate–EDTA buffer and ethidium bromide (0.5μg/ml).

An aliquot (1μl) of the PCR product was inserted into a pcDNA3.1 expression vector by the TA-cloning system and transformation was performed on Escherichia coli TOP10 cells following the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The selected transformant, CD38-pcDNA3.1, was analysed for presence and correct orientation of the insert by PCR and sequencing, grown in LB medium overnight and purified by the Quantum Prep plasmid midiprep kit (Biorad, Hercules, CA, USA).

CD38-pcDNA3.1 plasmid (20μg) was used to stable transfect LG14 and K562 cells by electroporation (Gregorini et al., 2002). After a 2-week incubation in medium containing 1mg/ml neomycin (G418, Invitrogen, Carlsbad, CA, USA), resistant colonies were isolated, cloned by limiting dilution, and referred to as LG14 CD38+ and K562 CD38+. Transfected cells, routinely screened for CD38 expression by IIF and flow cytometric analysis, showed >95% purity (Fig. 1B).

2.3 Oligonucleotide (ODN) treatment

CD38 expression was inhibited by blocking the gene promoter with antisense oligodeoxynucleotides (asODN): 5′-GCT GAA CTC GCA GTT GGC CAT-3′. Control sequences were non-sense oligodeoxynucleotides (nsODN) with a scrambled asODN sequence (5′-ACT CGG ATT CGG GTA CCT CAG-3′) and sense oligodeoxynucleotides (sODN) showing the same sequence and orientation as the target (5′-ATG GCC AAC TGC GAG TTC AGC-3′). ODNs' were modified by phosphorothiolation and synthesized by MWG-Biotech (Milan, Italy). The ODNs transfection was performed with oligofectamine reagent according to the manufacturer's protocol (Invitrogen). Cells were incubated with the ODNs/oligofectamine mixture for 24h, essayed for their CD38 expression by IIF and then exposed to apoptotic stimuli.

2.4 Apoptosis induction and evaluation

Unstimulated CD38+ and CD38 subpopulations showed apoptotic cell death in the range of 3–5%. In all cell lines, optimal concentrations and incubation times of each stimulus were selected by analysis of dose–response curves (Fig. 2A) and corresponded to those which induced apoptosis in at least 30% of CD38 cells.


Fig. 2

Dose-effect plots of H2O2 treatment on Raji CD38 cells. (A) Titration curve (H2O2 titred from 0.5 to 3mM); results are presented as the mean values±SD. (B and C) Annexin V-FITC/PI flow cytometry dot plots showing at which concentrations of H2O2 cells underwent apoptosis (1mM) or started necrosis (1.5mM), respectively. Apoptotic cells (Annexin V-FITC positive/PI negative) are in the lower right quadrant; necrotic cells (Annexin V-FITC positive/PI positive) are in the upper right quadrant. Similar experiments were performed on CD38 fractions of LG14, Jurkat and K562 cell lines.


2.4.1 H2O2 treatment

Hydrogen peroxide (H2O2) is a representative ROS (reactive oxygen species) and has been extensively used to study the apoptosis of cells following oxidative stress. Since H2O2 can induce both necrosis as well as apoptosis and its effect seems to be dosage-dependent, we assessed when cells underwent apoptosis or started undergoing necrosis (see Fig. 2B and C). Hence, cells (0.5×106/ml) were treated with H2O2 (Sigma, Milano, Italy) at the chosen concentration of 1mM and incubated at 37°C for 24h. After treatment, cells were collected, washed and analysed for apoptosis.

2.4.2 UV-B treatment

We used a Philips TL 20W/12 lamp calibrated routinely. The irradiation flux density to the cells was 2.2W/m2, as verified by an Osram centra UV meter. Prior to UV-B treatment, culture medium was removed, successively cells (0.5×106/ml) were irradiated and the medium was added again. Exposure times were determined, respectively, as 7min for LG14 and K562 and 3min for Jurkat and Raji cell lines. Cells were analysed 24h after UV irradiation.

2.4.3 α-TOS treatment

As widely reported, α-Tocopheryl Succinate (α-TOS), an esterified vitamin E analogue, shows a selective toxicity for malignant cells, is a potent inducer of apoptosis and anti-cancer agent. In our experiments, cells (0.5×106/ml) were treated by addition of 40μM (final concentration) α-TOS (Sigma), incubated at 37°C in a 5% CO2 atmosphere for 24h, then washed, collected and analysed.

2.4.4 hrTRAIL treatment

Cells (0.5×106/ml) were treated by addition of 50ng/ml (final concentration) tumour necrosis factor (TNF)-related apoptosis-inducing ligand (hrTRAIL), incubated at 37°C in a 5% CO2 atmosphere for 24h, then washed, collected and analysed.

Quantitative determination of apoptosis was performed by flow cytometric analysis with Annexin V-FITC and Propidium Iodide (PI) stained cells (Martin et al., 1996). Samples were analysed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). The number of apoptotic and necrotic cells was calculated using a computer program (Cell Quest Software, Becton Dickinson).

2.5 Statistical analysis

All experiments were conducted at least in triplicates and data are shown as mean±SD. Statistical significance (p<0.05) of differences between CD38+ and CD38 subpopulations' susceptibility to apoptosis was determined by a two-way replicate analysis of variance (ANOVA).

3 Results

Two subpopulations (CD38+ and CD38) of each cell line (LG14, Raji, Jurkat and K562), sorted by their CD38 expression, were treated with different apoptotic triggers (H2O2, UV-B, α-TOS, hrTRAIL). After 24h each fraction was analysed by the Annexin V-FITC/PI flow cytometric assay to measure the percentage of cells undergoing apoptosis (Annexin V-FITC positive/PI negative) (see Fig. 3).


Fig. 3

Representative examples of two parameter (Annexin V-FITC/PI) flow cytometry dot plots showing the effect of UV-B (3min exposure at 2.2W/m2), H2O2 (1mM), α-TOS (40μM) and hrTRAIL (50ng/ml) on apoptosis in Raji CD38+ cells (0.5×106cells/ml). Percentages of apoptotic cells are indicated in each lower right quadrant. The CONTROL dot plot refers to Raji CD38 cells incubated with H2O2 (1mM) for 24h; analogous apoptotic percentages were obtained for Raji CD38 cells treated with UV-B, α-TOS and hrTRAIL at the concentrations indicated above. Analyses of CD38+ and CD38 fractions of Jurkat, LG14 and K562 cell lines provided similar results (for details see Section 2 and Fig. 4).


Our data revealed that, irrespective of the trigger used, CD38 expression significantly enhanced the LG14, Raji and Jurkat cells' propensity to enter the apoptotic pathway; indeed, compared to CD38, lymphoma CD38+ cells were particularly responsive to apoptotic stimulation. On the contrary, CD38 expression had no effect on K562 cells, whose CD38+ subpopulation was as prone to apoptosis as the CD38 one (Fig. 4).


Fig. 4

Apoptosis induction in CD38+ and CD38 lymphoma and myeloid leukemia cells. Lymphoma B cells (LG14 and Raji), lymphoma T cells (Jurkat) and myeloid erythroleukemia cells (K562) were sorted for their CD38 expression and treated with H2O2 (1mM), UV-B (irradiation flux of 2.2W/m2; 7min exposure time for LG14 and K562, 3min for Jurkat and Raji), α-TOS (40μM) and hrTRAIL (50ng/ml). After 24h, apoptosis was evaluated by Annexin V-FITC/PI flow cytometry assay and expressed as percentage (%). All data are reported as mean±SD from three independent experiments. “*” indicates statistical differences (p<0.05) in the values for CD38+ and CD38 cells.


In lymphoma cells, suppression of CD38 gene transcription invariably reduced apoptotic death of CD38+ cells to the same extent as that observed in CD38 subpopulations (compare asODN bars of LG14, Raji and Jurkat in Fig. 5 with corresponding CD38 bars in Fig. 4). Non-sense ODN and sense ODN, which do not affect CD38 gene expression, were used as controls. As an expected result, the propensity to apoptotic death of CD38+ cells previously treated with nsODN or sODN always matched with that of ODNs-untreated CD38+ cells (compare nsODN and sODN bars of LG14, Raji and Jurkat in Fig. 5 with corresponding CD38+ bars in Fig. 4). Finally, antisense-treated K562 CD38+ cells showed nearly the same percentage of apoptotic events as in ODNs-untreated K562 CD38 and CD38+ cells (compare asODN bars of K562 in Fig. 5 with K562 CD38 and CD38+ bars in Fig. 4). The same ODNs treatment (using asODN, nsODN and sODN) was applied to CD38 subpopulations of each cell line. Obtained results (data not shown) were not different from those observed in ODNs-untreated CD38 cells.


Fig. 5

Apoptosis induction in CD38+ lymphoma and myeloid leukemia cells after antisense oligonucleotide (asODN) treatment. CD38+ lymphoma (LG14, Raji and Jurkat) and myeloid leukemia (K562) cells were treated with antisense (asODN) oligonucleotides targeting CD38 and then exposed to H2O2, UV-B, α-TOS and hrTRAIL at the concentrations indicated in Section 2. Non-sense (nsODN) and sense (sODN) oligonucleotides were used as experimental controls. Apoptosis was evaluated by Annexin V-FITC/PI assay and expressed as a percentage (%). All data are reported as mean±SD from three independent experiments and “*” indicates statistical differences, p<0.05.


4 Discussion

In the present study, we demonstrate that, at least in T and B lymphoma cells, CD38 expression modulates the propensity of CD38+ fractions to undergo apoptosis and that its effect is independent of the stimuli used. Inhibition of CD38 by antisense oligonucleotides before apoptotic stimulation resulted in a decreased rate of cell death, proving that enhanced propensity to apoptosis observed in CD38+ cells was strongly associated with CD38 expression. This finding confirms the existence of a correlation between expression of CD38 and susceptibility to apoptotic challenges, previously suggested by several authors (e.g. Zupo et al., 1996; Tenca et al., 2003). Moreover, the occurrence of nearly the same percentages of dead cells both in biochemical and receptor-mediated apoptosis leads us to suggest that CD38 could exploit more than one of the cellular signalling pathways involved in apoptosis induction, which, on the other hand, would fit with its “parasitic” attitude and structural and functional evolutive conservation (Deaglio et al., 2001).

Data concerning K562 are still unclear. Indeed K562 CD38+ cells did not show any enhanced propensity to apoptosis, compared with K562 CD38 under the same experimental conditions. In this regard, taking into account that myeloid K562, unlike lymphoma cells, do not constitutionally express CD38 (since they are not involved either in immunological events or in cytotoxic reactions), it is conceivable that naïve K562 lack a CD38 pathway, and this might explain the absence of susceptibility to apoptosis even in CD38-transfected cells.

Finally, it is noteworthy that the correlation between CD38 expression levels and cells susceptibility to apoptosis makes this molecule a valuable prognostic factor in leukemia. Particularly in acute adult leukemias (AML and ALL), increased CD38 expression is associated with a favourable prognosis (Keyhani et al., 2000). On the contrary, in B-cell chronic lymphocytic leukemia (B-CLL), CD38+ patients are characterised by an unfavourable clinical course (Ibrahim et al., 2001; Morabito et al., 2002; Matrai, 2005). Hence, it could be argued that, in B-CLL, CD38 expression seems to have quite an opposite effect. Nevertheless, a possible explanation of such an apparent contradiction might be the creation of a network between CD38 and other surface receptors leading to B-CLL cell growth and survival (Deaglio et al., 2005).

Although our observations add new insights to the understanding of CD38 functions, its biological role remains far from being fully disclosed and more investigations will be required to uncover its elusive nature.

Acknowledgments

We wish to thank Dr R. Coles (University of Urbino, Italy) for English language revision and two anonymous reviewers whose comments and suggestions substantially improved the manuscript.

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Received 15 February 2006/11 April 2006; accepted 10 May 2006

doi:10.1016/j.cellbi.2006.05.004


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)