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Cell Biology International (2006) 30, 727732 (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
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 (H
Keywords: CD38, Apoptosis, Lymphoma T cells, Lymphoma B cells, Myeloid leukemia cells.
*Corresponding author. Tel.: +39 722 303438; fax: +39 722 303436.
Human CD38 is a 42–45
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 H
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, 2
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).
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′
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 5
An aliquot (1
CD38-pcDNA3.1 plasmid (20
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 24
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.
Dose-effect plots of H
Hydrogen peroxide (H
2.4.2 UV-B treatment
We used a Philips TL 20W/12 lamp calibrated routinely. The irradiation flux density to the cells was 2.2
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
2.4.4 hrTRAIL treatment
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
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 (H
Representative examples of two parameter (Annexin V-FITC/PI) flow cytometry dot plots showing the effect of UV-B (3
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).
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 H
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.
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 H
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.
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 2006doi:10.1016/j.cellbi.2006.05.004