Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation

Cell lines used in this study were obtained from DSMZ (Braunschweig, Germany), ATCC (Manassas, VA), ECACC (Porton Down, UK) or from the originators (see Additional file 1: Table S1). Authenticity check of cell lines was performed by short tandem repeat analysis by the commercial providers. Early passage cells were cultured in suspension in the appropriate culture medium at 37 °C in humidified cell culture incubators in a 95% air 5% CO₂ atmosphere. Fetal calf serum that supported vigorous growth of the Kasumi-2 line as single cells without clumping was selected for culture. Mycoplasma screening was performed by polymerase chain reaction using the G238 mycoplasma detection kit of Applied Biological Materials, Euromedex, Souffelweyersheim, France. Cell densities and viabilities of exponentially growing cultures were determined by cell counting using a Malassez chamber and trypan blue exclusion.

Before treatments cells were centrifuged and suspended in new complete medium at densities indicated in Figures and distributed into 60 cm² plastic Petri dishes. Non-adherent dishes were used for the various cell lines because treatment in adherent conditions resulted in the adherence of a minor fraction (<5%) of the cells following treatments, for example for Kasumi-2 cells. Treatment of MHH-CALL3 cells was done in cell culture grade adherent Petri dishes, since treatment of this cell line leads to the adherence of the overwhelming majority (>90%) of viable cells.

When PMA treatments were applied in the presence of Gö 6983 or GF 109203X, these were added to the cell cultures 30 min before the addition of PMA. PMA and thapsigargin in double treatment experiments were added simultaneously. Drugs were thereafter present during the entire experiment.

After the appropriate treatments, cells were harvested by centrifugation and washed with ice cold 150 mM NaCl. Total cellular protein was then precipitated with 5% trichloroacetic acid (TCA), centrifuged and dissolved in modified SDS PAGE lysis buffer. This sample preparation process, as well as quantification of the protein content of lysates was performed as described in [35]. Equal amounts of total cellular protein (60 μg/well) were applied to polyacrylamide gels, and SDS PAGE followed by immunoblotting onto nitrocellulose was done as previously described [35]. After electrotransfer, identical protein loading was checked by Ponceau red staining and densitometry [35].

Western blotting for SERCA2 and SERCA3 was performed using the IID8 (Sigma-Aldrich, 0.4 μg/ml) and the PLIM430 mouse monoclonal (10x diluted hybridoma cell culture supernatant) antibodies, respectively, and peroxidase-conjugated anti-mouse Ig antibodies as in [35]. Luminescent signal obtained with the ECL Prime (Amersham) and SuperSignal West Femto Maximum Sensitivity (Thermo Fisher) reagents was detected, quantified and analyzed using a BioRad ChemiDoc MP image acquisition system and the Image Lab 4.0 software. Immunoblot signal intensities were normalized to the corresponding total protein values as obtained by Ponceau red staining, and are expressed as fold change in treated cells versus untreated control.

Detection by Western blotting of CD20 (clone L26 purified mouse monoclonal anti-human CD20cy, Dako Denmark A/S, 0.2 μg/ml), RAG-1 (Santa Cruz Biotechnology, sc-5599, H-300, rabbit polyclonal IgG 0.2 μg/ml), TdT (clone EPR2976Y, rabbit hybridoma culture supernatant monoclonal antibody, dilution:3500x, Epitomics), CD19 (clone LE-CD19 purified mouse monoclonal anti-human CD19, Thermo Fisher Scientific, 0.33 μg/ml) was performed similarly.

Detection and analysis of expression of various lymphoid phenotypic markers (CD3, CD5, CD10, CD19, CD20, CD22, CD34, CD38, CD45, FMC7, TdT, κ and λ light chains and IgM) by flow cytometry was done as previously described [36, 37].

Immunocytochemical staining for CD20 expression was performed on cytologic smears. Suspensions of treated and untreated control cells of packed cell volume ratio of approximately 50% were applied to poly-lysine coated microscopic slides and air-dried overnight. Following fixation in acetone at room temperature for 10 min and drying the slides were rehydrated and labeled for CD20 expression using the Clone L26 monoclonal mouse anti-CD20 antibody (Dakocytomation, Les Ulis, France) at a concentration of 6 μg/ml in Dako REAL^TM antibody diluent (Dakocytomation), using an indirect avidin-biotin-peroxidase method with 3,3’diaminobenzidine (DAB) as chromogen on an automated immunostainer (Benchmark®, Ventana Medical Systems, Illkirch, France). Endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide in phosphate-buffered saline for 10 min. Incubation with the CD20-specific antibody was carried out at 37 °C for 30 min, and labeling was revealed using the Ventana iView® DAB Detection kit with copper enhancement, according to the instructions of the manufacturer. Slides were counterstained with hematoxylin and bluing reagent (Ventana). Cells were photographed using a Zeiss Axio Scope.A1 microscope and an AxioCam ICc1 camera using the Axiovision 4.8.2 software. May-Grünwald-Giemsa staining of cytological smears was done using standard methods.

Following treatments cells were centrifuged, and RNA was extracted with the RNeasy Mini Kit of Qiagen (Courtaboeuf, France). RT-PCR was performed on 1 μg of total RNA using the Iscript cDNA synthesis kit of Bio-Rad (Marne la Coquette, France) TaqMan gene expression assays were performed on an ABI prism 7000 apparatus (Applied Biosystems, Courtaboeuf, France) with the Hs 99999905_m1, Hs 0054487_m1 and Hs 01024558_m1 primer pairs (Applied Biosystems, Life Technologies, Fisher Scientific, Illkirch, France) for GAPDH, ATP2A2 (SERCA2) and ATP2A3 (SERCA3), respectively. Relative gene expression levels after treatments were calculated by the 2^-ΔΔCt method normalized to GAPDH, and expressed as fold change compared to untreated cells [38].

As investigated in the Kasumi-2 and RCH-ACV cell lines that carry the t(1;19)(q23;p13) translocation and express the E2A-PBX1 fusion oncoprotein, PMA treatment led to enhanced SERCA3 expression. This could be observed from 10⁻¹⁰-10⁻⁹ M PMA, and reached a plateau in the 10⁻⁸-10⁻⁷ concentration range (Fig. 1a and b). Induction of SERCA3 expression was also manifested on the mRNA level. As shown in Fig. 1c and d, induction of SERCA3 mRNA expression was observed in both Kasumi-2 and RCH-ACV cells, already at 12 h of treatments, and this followed a reproducible biphasic pattern with a 5-6-fold enhancement when compared to untreated control. Moreover, the moderate enhancement of SERCA2 protein expression observed in Kasumi-2 cells could also be observed on the mRNA level.

Significant induction of SERCA3 protein expression was observed starting at day 1-2 and reached a plateau at day 4-5 (Fig. 2a and b). Maximal induction in the cell lines was approximately five- to sevenfold when compared to untreated cells. DMSO vehicle when applied alone was without effect. As shown in Fig. 2c and d, PMA treatment in Kasumi-2 cells led to inhibition of cell proliferation (Panel C) with maintained viability (Panel D) up till day 5, whereas longer treatments led to significant cell death.

SERCA3 expression was induced by PMA in all available precursor B ALL cell lines bearing the t(1;19)/E2A-PBX1 translocation. As shown in Fig. 3, the Kasumi-2, RCH-ACV, LK63, Lila-1, MHH-CALL3 and 697 cell lines all simultaneously expressed SERCA2 and SERCA3 calcium pump isoforms. When the cells were treated with 10⁻⁷ M PMA for 5 days, the expression of SERCA3 protein was strongly enhanced in all cell lines, whereas expression of the SERCA2 protein remained unchanged or was slightly modified in a cell-line dependent manner. This indicates that the induction of SERCA3 expression is a universal phenomenon in all E2A-PBX1 expressing cell lines investigated in this study. On the other hand, we found that PMA treatment of precursor B ALL cell lines (Nalm-21, TOM-1, BV173, KOPN-8 and Reh) of other molecular types (see Additional file 1: Table S1) led to the death of cultures within 24–48 h.

The phenotype of Kasumi-2 precursor B ALL cells was investigated before and after stimulation with 10⁻⁷M PMA for 5 days using established markers of early B cell differentiation. Whereas before treatment cells expressed high levels of TdT and RAG-1 and low levels of CD20 proteins, PMA treatment led to strong CD20 expression and to the simultaneous down-regulation of TdT and RAG-1 expression (Fig. 4a).

PMA-treatment of cells led to the formation of multicellular aggregates and to morphological changes similar to a plasmacytoid morphology (Fig. 4b). Whereas untreated cells were round, small and displayed high nucleocytoplasmic ratio and round nuclei, treatment with 10⁻⁸ M PMA for 4 days led to increased cell size, more abundant, often excentral cytoplasm, lower nucleocytoplasmic ratio and frequently bilobed nuclei with clear paranuclear areas.

As studied by flow cytometry, and similarly to previous observations made on the LK63 and Lila-1 t(1;19) cell lines [39], untreated Kasumi-2 cells displayed a precursor B lymphoid phenotype (CD3⁻, CD5⁻, CD10⁺, CD19⁺, CD20^+/-, CD22⁺, CD34⁻, CD38⁺, CD45⁺, TdT⁺). As shown in Fig. 5, treatment of cells with 10⁻⁸ M PMA for 4 days led to markedly enhanced expression of the CD19, CD20, CD22 and FMC7 differentiation markers, whereas the expression of markers of immaturity such as CD10 or TdT was decreased. PMA treatment, however, did not lead to κ or λ chain or IgM expression (not shown), indicating however, that the cells do not attain a mature B cell phenotype.

The induction of SERCA3 expression by PMA was investigated in the presence or absence of pharmacological inhibitors of PKC. As shown in Fig. 6, when cells were preincubated with Gö 6983 or with GF 109203X for 30 min, the consecutive addition of 10⁻⁸ M PMA (a maximally active concentration) was without effect on SERCA expression, indicating the involvement of conventional or novel-type PKC activity in PMA-induced SERCA expression.

In order to further correlate SERCA3 expression with the state of differentiation of neoplastic B cells, SERCA3 expression of precursor B cell lines was compared to that of an extensive set of randomly selected mature B cell lines (see Additional file 1: Table S1). As shown in Fig. 7, SERCA3 expression in all individual precursor B cell lines was weaker than in any of the investigated mature cell lines. A highly significant, on average 15-fold higher SERCA3 expression could be observed in mature B cell lines when compared with the average expression in the precursor B cell group.

The role of SERCA function in the differentiation of leukemic B cells was investigated using thapsigargin, a high affinity, selective inhibitor of SERCA-dependent calcium transport [40]. As shown in Fig. 8, Treatment of Kasumi-2 cells with sub-nanomolar concentrations of thapsigargin completely abolished the induction of CD20 expression induced by PMA (Fig. 8a). Inhibition of PMA-induced CD20 expression by thapsigargin was observed in the absence of significant toxicity during the experiments (Fig. 8b), although higher thapsigargin concentrations were, in accordance with the literature [41], toxic. Similar observations were made with cyclopiazonic acid or 2,5-di-tert-butyl-1,4-benzohydroquinone, two other, structurally unrelated SERCA inhibitors as well (data not shown).