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Positive regulation of deoxycytidine kinase activity by phosphorylation of Ser-74 in B-cell chronic lymphocytic leukaemia lymphocytes
Cancer Letters 253 (2007) 68–73 www.elsevier.com/locate/canlet
Positive regulation of deoxycytidine kinase activity by phosphorylation of Ser-74 in B-cell chronic lymphocytic leukaemia lymphocytes Caroline Smal a, Eric Van Den Neste a,b,*, Marie Maerevoet b, Xavier Poire´ b, Ivan The´ate c, Franc¸oise Bontemps a a
Laboratory of Physiological Chemistry, Christian de Duve Institute of Cellular Pathology and Universite´ catholique de Louvain, B-1200 Brussels, Belgium b Department of Hematology, Cliniques universitaires Saint-Luc, Universite´ catholique de Louvain, B-1200 Brussels, Belgium c Department of Pathology, Cliniques universitaires Saint-Luc, Universite´ catholique de Louvain, B-1200 Brussels, Belgium Received 8 November 2006; accepted 15 January 2007
Abstract Deoxycytidine kinase (dCK) activates several antileukaemic nucleoside analogues. We have recently reported that the activity of dCK, overexpressed in HEK 293T cells, correlates with its phosphorylation level on Ser-74. Here, we show that dCK from B-cell chronic lymphocytic leukaemia (B-CLL) lymphocytes can be detected by an anti-phospho-Ser-74 antibody and that interindividual variability in dCK activity is related to its phosphorylation level on Ser-74. Moreover, pharmacological intervention modified Ser-74 phosphorylation, in close parallel with changes in dCK activity. These results suggest that activation of dCK via phosphorylation of Ser-74 might constitute a new therapeutic strategy to enhance activation and efficacy of nucleoside analogues. 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: B-CLL lymphocytes; Deoxycytidine kinase; Immunocytochemistry; Nucleoside analogues; Ser-74 phosphorylation
1. Introduction Phosphorylation of chemotherapeutically important nucleoside analogues, such as fludarabine
*
Corresponding author. Address: Department of Hematology, Cliniques universitaires Saint-Luc, Universite´ catholique de Louvain, B-1200 Brussels, Belgium. Tel.: +32 27647568; fax: +32 27647598. E-mail address: [email protected] (E. Van Den Neste).
(9-b-D-arabinosyl-2-fluoroadenine) or CdA (2-chloro-2 0 -deoxyadenosine), by deoxycytidine kinase (dCK; EC 2.7.1.74) is a prerequisite for their pharmacological action [1]. Identification of mechanisms controlling dCK activity is of particular interest because down-regulation of this enzyme can decrease chemosensitivity to nucleoside analogues in diseases such as B-cell chronic lymphocytic leukaemia (B-CLL) [2–4]. We, and others, have shown that agents known to modify the phosphorylation status of proteins can modulate dCK activity
0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.01.013
C. Smal et al. / Cancer Letters 253 (2007) 68–73
in various types of leukaemic cells, including B-CLL lymphocytes [5–7]. Recently, we have identified by mass spectrometry multiple phosphorylation sites in dCK after overexpression in HEK 293T cells, the most abundant of these being Ser-74. Site-directed mutagenesis demonstrated that Ser-74 phosphorylation was crucial for dCK activity in the latter model [8]. The purpose of the present study was to explore phosphorylation of dCK on Ser-74 in lymphocytes from patients with B-CLL, and its relation to dCK activity. 2. Materials and methods 2.1. Materials Ficoll–Paque Plus (density: 1.077), [5-3H]-deoxycytidine (21 Ci/mmol), Hybond C-extra membranes, ECL enhanced chemiluminescence kit were from GE Healthcare. FCS and penicillin–streptomycin were purchased from BioWhittaker Europe. RPMI-1640 and all tissue culture reagents were from Gibco/Invitrogen. Anti-dCK antibody raised against the C-terminal peptide of human dCK (amino acids 246–260) was generated by Eurogentec, according to the procedure of Hatzis et al. [9]. Anti-phospho-Ser-74 antibody was obtained as described in [8]. Horseradish peroxidase conjugated anti-rabbit antibody was purchased from Sigma–Aldrich. CdA was synthesised and supplied by Prof. J. Marchand (Laboratory of Organic Chemistry, Universite´ catholique de Louvain, Louvain-la-Neuve). Stock solutions of CdA were prepared in ethanol/150 mM NaCl (v/v). Other chemicals, materials and reagents were from Sigma, Merck Biosciences or Bio-Rad Laboratories. 2.2. Cell isolation and incubation Freshly obtained peripheral blood from B-CLL patients was fractionated by Ficoll–Paque sedimentation. Mononuclear cells (P95% B-CLL cells, as confirmed by flow cytometry analysis) were washed and directly lysed, or resuspended in RPMI-1640, supplemented with 10% FCS and 1% penicillin–streptomycin, and incubated at 37 C in 5% CO2 in air. All patients had an established diagnosis of B-CLL, were free of any anticancer treatment for at least 3 months, had lymphocytes >30 · 109/L, and had given informed consent according to institutional guidelines [10]. 2.3. dCK assay and immunoblotting Cell lysis and dCK assay with 10 lM deoxycytidine and 5 mM MgATP as substrates were performed as described in [5]. Immunoblotting with anti-dCK antibody or anti-phospho-Ser-74 antibody was carried out as
69
reported in [8]. dCK expression or phosphorylation were quantified by using National Institutes of Health image software. 2.4. Immunocytochemistry The cells (105 cells/cytospin) were cytocentrifuged and fixed in 10% formol for 10 min. Endogenous peroxidases were blocked (peroxidase blocking kit-DAKO), and non-specific antibody staining was prevented by preincubation in 10% normal goat serum for 30 min. Cytospins were then incubated with the phosphospecific antibody (diluted 1/5), after purification by affinity chromatography on phosphopeptide-coupled thiol Sepharose resin (GE Healthcare), as previously described [11]. They were revealed using the Envision system (DAKO) with diaminobenzidine (Sigma–Aldrich). The samples were counterstained using hematoxylin, and examined under light microscopy. 2.5. Statistical analysis The statistical analysis was performed using the twotailed Student’s t-test and the Pearson correlation coefficient. Statistical significance was considered when P values were less than 0.05. 3. Results and discussion We first sought to investigate whether dCK was phosphorylated on Ser-74 in B-CLL cells by probing cell lysates with the anti-phospho-Ser-74 antibody. Our previous work had demonstrated that this antibody is specific for a phosphoresidue in that it fails to detect overexpressed dCK in HEK 293T cell lysates treated with k-protein phosphatase [8]. As illustrated in Fig. 1a (upper panel), a signal is detected with the anti-phospho-Ser-74 antibody at the dCK level. As it is suppressed in the presence of an excess of the phosphopeptide used to generate the antibody (not illustrated), we can conclude that it really corresponds to a phosphorylation of Ser-74. These results accord with our findings in CCRF-CEM cells [8]. Interestingly, the level of phosphorylation of dCK on Ser-74 was variable amongst patients, ranging from almost undetectable (lanes 1, 5, 6, 9) to strong signals (lanes 2, 3, 7, 8). Probing with an anti-dCK antibody (Fig. 1a, lower panel) showed that the amount of dCK protein also varied amongst patients, and was not strictly correlated with the level of phosphorylation of dCK. Indeed, patients with relatively similar levels of total dCK (i.e., lanes 2, 3, 4, 6, 8) displayed considerably different states of Ser-74 phosphorylation. As given in Fig. 1a, values of dCK activity also displayed broad variations between patients. After quantification of dCK phosphorylation and dCK level by densitometric analysis in samples of 15 different patients, dCK activity was plotted against phospho-dCK/total dCK ratio (Fig. 1b) or
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C. Smal et al. / Cancer Letters 253 (2007) 68–73
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Patients 1
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pSer-74-dCK (arbitrary units) Fig. 1. dCK phosphorylation on Ser-74 in B-CLL lymphocytes and its relation to dCK activity. (a) Lysates (100 lg) of lymphocytes from ten B-CLL patients were subjected to SDS–PAGE followed by immunoblotting with the anti-phospho-Ser-74 antibody (upper part), or with the anti-dCK antibody as a control for dCK expression (lower part). dCK activity, expressed in pmol/min/mg of protein, is given below each corresponding patient. (b) dCK activity was plotted against phospho-dCK/total dCK ratio, assessed by densitometric quantification, in 15 B-CLL samples (including in addition the five control samples of Fig. 2). Analysis by linear least squares regression gave r = 0.90 with P < 0.0001. (c) dCK activity was plotted against phospho-dCK and analyzed as in b (r = 0.95; P < 0.0001).
against phospho-dCK alone (Fig. 1c). Analysis by linear least squares regression showed a significant positive correlation between dCK activity and both phospho-dCK/total dCK ratio (r = 0.90) or phospho-dCK (r = 0.95), indicating that the phosphorylation state of dCK plays
an important role in the level of basal dCK activity in BCLL cells. It is interesting to note that the correlation between dCK activity and dCK phosphorylation is better than the correlation previously established between dCK activity and dCK content (r = 0.554) [12].
C. Smal et al. / Cancer Letters 253 (2007) 68–73
Our next objective was to explore whether the dCK Ser-74 phosphorylation level could be modified in BCLL cells, and whether this modification would correlate with changes in dCK activity. For this purpose, B-CLL cells were treated with various agents able to increase or decrease dCK activity [5–8,13,14]. As shown in Fig. 2 (patients 11–13 and 15), CdA, etoposide (VP-16), UV-C irradiation, genistein (GNT) and aphidicolin (APC) all increased dCK phosphorylation on Ser-74, in close parallel with dCK activity. These results strongly suggest that all the agents tested increase dCK activity via phosphorylation of Ser-74. Conversely, hyperosmotic stress induced by sorbitol (SORB) significantly reduced Ser-74 phosphorylation, as well as dCK activity in BCLL lymphocytes (patient 15), as previously observed in dCK-overexpressing HEK 293T cells [8]. Fig. 2 also illustrates a sample (patient 14) in which SORB did not significantly reduce dCK activity, probably because basal phosphorylation of dCK was already at a very low level. In each patient, expression of total dCK remained unchanged in all conditions tested. Taken together, our results validate the previous hypothesis that dCK activity is regulated by reversible phosphorylation in leukaemic cells [5,6,8,13,14], although other mechanisms besides phosphorylation might also be involved in the control of this enzyme activity [15]. Indeed, in patient 12, there is clearly a close parallellism between increase in Ser-74 phosphorylation and dCK activity, but the values of dCK activity after treatment are lower than in patients 11 and 13, despite an apparently higher ratio phosphodCK/total dCK.
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Finally, we performed immunocytochemistry with purified anti-phospho-Ser-74 antibody in both CCRFCEM and B-CLL cells to analyze whether increase of Ser-74 phosphorylation could be detected in situ and could modify subcellular localization of phospho-dCK (Fig. 3). No staining of CCRF-CEM cells was detected in the absence of the primary anti-phospho-antibody (Fig. 3a). In contrast, cytoplasmic staining was seen with the anti-phospho-Ser-74 antibody (Fig. 3b), confirming recent results with an antibody directed against the C-terminal end of the dCK protein [16]. Moreover, staining was increased after incubation with VP-16 (Fig. 3c), which activates dCK in this cell line [13]. The specificity of the staining was ensured by adding an excess of the phosphopeptide used to raise anti-Ser-74 antibodies in rabbit, which almost completely abolished immunostaining (Fig. 3d). Phospho-dCK was also located in the cytoplasm of most of control B-CLL lymphocytes, with a tendency to scatter in perinuclear areas (Fig. 3e). After incubation with CdA (Fig. 1f), VP-16 (Fig. 3g), or after UV irradiation (Fig. 3h), most B-CLL lymphocytes examined were also positive for phospho-Ser-74 dCK, but with a greater intensity of staining with respect to control cells. The immunostaining of treated cells had a granular appearance mainly expressed in perinuclear and submembrane regions, which is not in favor of a translocation of dCK from the cytoplasm into the nucleus when it is activated by phosphorylation. In the same samples, we confirmed that dCK activity (see insets of Fig. 3b–h) and dCK phosphorylation on Ser-74 (not shown) were increased by CdA, VP-16 or UV-light.
Fig. 2. Phosphorylation of dCK on Ser-74 in relation with changes in dCK activity. B-CLL lymphocytes were incubated without () or with 1 lM CdA, 100 lM etoposide (VP-16), 100 lM genistein (GNT) or 10 lM aphidicolin (APC) for 2 h, or with 0.5 M sorbitol (SORB) for 1 h, or UV-C-radiated (30 J/m2) and thereafter incubated for 30 min (or 2 h for patient 13). Cell lysates (50–100 lg) were analysed for dCK phosphorylation on Ser-74 and dCK content as in Fig. 1a, and for dCK activity.
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Fig. 3. Immunocytochemical staining of dCK phosphorylated on Ser-74 in CCRF-CEM (a–d) or in B-CLL lymphocytes (e–h). Cytospins were prepared and stained with anti-phospho-Ser-74 antibody as described in Section 2. (a) Negative control. (b) Untreated CCRF-CEM cells. (c) CCRF-CEM cells treated with 100 lM VP-16 for 2 h. (d) Same as in (c) except that the anti-phospho-Ser-74 antibody was blocked with the antigenic phosphopeptide. (e) Untreated B-CLL cells. (f) B-CLL cells treated with 1 lM CdA for 2 h. (g) B-CLL cells treated with 100 lM VP-16 for 2 h. (h) B-CLL cells incubated for 30 min after UV-C radiation (30 J/m2). dCK activity (pmol/min/mg protein) was determined in parallel samples (insets). Images were acquired using a Zeiss Axioplan microscope (Zeiss, Go¨ttingen, Germany) with a 40· Plan Neofluar objective (NA 0.75). Images were captured with a HRc CDD camera (Axiocam, Zeiss), and processed using Axiovision software (Zeiss).
We conclude that phosphorylation of dCK on Ser-74 is crucial for dCK activity in B-CLL cells. This accords with our previous finding that mutation of Ser-74 to a nonphosphorylatable residue strongly decreased human dCK activity overexpressed in HEK 293T cells [8]. This post-translational mechanism of regulation is likely to be implicated in the high variability of dCK activity amongst B-CLL patients, which is not satisfactorily explained by changes in dCK mRNA levels [17]. It should be noted, however, that other mechanisms of dCK inactivation have been reported in patients with acute myeloblastic leukaemia (AML), including alternatively spliced dCK mRNA transcripts [18], or single nucleotide polymorphisms in the promoter region of dCK [19]. Mutation in the dCK gene rarely occurs in AML patients [20–22]. Finally, because phosphorylation of dCK on Ser-74 can be increased by pharmacological intervention, our results may reinforce strategies aimed at restoring or increasing dCK activity to improve clinical efficacy of nucleoside analogues in B-CLL [23,24]. Indeed, uptake of nucleoside analogues, which includes transport through the plasma membrane, but also their phosphorylation, may be reduced in some patients, causing nucleoside analogue resistance [3]. Studies are in progress to assess the relationship between phosphorylation of dCK on Ser-74, activation of various nucleoside analogues, and in vitro sensitivity to the latter drugs.
Acknowledgements This work was supported by grants from the Belgian Fonds National de la Recherche Scientifique (Te´le´vie, FRSM, Cre´dit au Chercheur), by the Interuniversity Attraction Poles Program-Belgian Science Policy (P5/05), and by private foundations: Bauchau, Maisin, Goor and Salus Sanguinis. We thank Professor G. Van den Berghe for helpful comments. References [1] E.S. Arner, S. Eriksson, Mammalian deoxyribonucleoside kinases, Pharmacol. Ther. 67 (1995) 155–186. [2] H. Kawasaki, C.J. Carrera, L.D. Piro, A. Saven, T.J. Kipps, D.A. Carson, Relationship of deoxycytidine kinase and cytoplasmic 5 0 -nucleotidase to the chemotherapeutic efficacy of 2-chlorodeoxyadenosine, Blood 81 (1993) 597–601. [3] C.M. Galmarini, J.R. Mackey, C. Dumontet, Nucleoside analogues: mechanisms of drug resistance and reversal strategies, Leukemia 15 (2001) 875–890. [4] J.R. Mackey, C.M. Galmarini, K.A. Graham, A.A. Joy, A. Delmer, L. Dabbagh, et al., Quantitative analysis of nucleoside transporter and metabolism gene expression in chronic lymphocytic leukemia (CLL): identification of fludarabinesensitive and -insensitive populations, Blood 105 (2005) 767–774. [5] C. Smal, S. Cardoen, L. Bertrand, A. Delacauw, A. Ferrant, G. Van den Berghe, et al., Activation of deoxycytidine
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[16] I. Hubeek, G.J. Peters, A.J. Broekhuizen, I. Talianidis, J. Sigmond, B.E. Gibson, et al., Immunocytochemical detection of deoxycytidine kinase in haematological malignancies and solid tumours, J. Clin. Pathol. 58 (2005) 695– 699. [17] K. Lotfi, K. Karlsson, A. Fyrberg, G. Juliusson, V. Jonsson, C. Peterson, et al., The pattern of deoxycytidine- and deoxyguanosine kinase activity in relation to messenger RNA expression in blood cells from untreated patients with B-cell chronic lymphocytic leukemia, Biochem. Pharmacol. 71 (2006) 882–890. [18] M.J. Veuger, M.W. Honders, J.E. Landegent, R. Willemze, R.M. Barge, High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia, Blood 96 (2000) 1517–1524. [19] J.Y. Shi, Z.Z. Shi, S.J. Zhang, Y.M. Zhu, B.W. Gu, G. Li, et al., Association between single nucleotide polymorphisms in deoxycytidine kinase and treatment response among acute myeloid leukaemia patients, Pharmacogenetics 14 (2004) 759–768. [20] M.M. van den Heuvel-Eibrink, E.A. Wiemer, M. Kuijpers, R. Pieters, P. Sonneveld, Absence of mutations in the deoxycytidine kinase (dCK) gene in patients with relapsed and/or refractory acute myeloid leukemia (AML), Leukemia 15 (2001) 855–856. [21] M.J. Veuger, M.W. Honders, R. Willemze, R.M. Barge, Deoxycytidine kinase expression and activity in patients with resistant versus sensitive acute myeloid leukemia, Eur. J. Haematol. 69 (2002) 171–178. [22] C.M. Galmarini, X. Thomas, K. Graham, A. El Jafaari, E. Cros, L. Jordheim, et al., Deoxycytidine kinase and cN-II nucleotidase expression in blast cells predict survival in acute myeloid leukaemia patients treated with cytarabine, Br. J. Haematol. 122 (2003) 53–60. [23] R.M. Mohammad, F.W. Beck, K. Katato, N. Hamdy, N. Wall, A. Al-Katib, Potentiation of 2-chlorodeoxyadenosine activity by bryostatin 1 in the resistant chronic lymphocytic leukemia cell line (WSU-CLL): association with increased ratios of dCK/5 0 -NT and Bax/Bcl-2, Biol. Chem. 379 (1998) 1253–1261. [24] I. Ahmad, A.M. Al-Katib, F.W. Beck, R.M. Mohammad, Sequential treatment of a resistant chronic lymphocytic leukemia patient with bryostatin 1 followed by 2-chlorodeoxyadenosine: case report, Clin. Cancer Res. 6 (2000) 1328– 1332.
Positive regulation of deoxycytidine kinase activity by phosphorylation of Ser-74 in B-cell chronic lymphocytic leukaemia lymphocytes Caroline Smal a, Eric Van Den Neste a,b,*, Marie Maerevoet b, Xavier Poire´ b, Ivan The´ate c, Franc¸oise Bontemps a a
Laboratory of Physiological Chemistry, Christian de Duve Institute of Cellular Pathology and Universite´ catholique de Louvain, B-1200 Brussels, Belgium b Department of Hematology, Cliniques universitaires Saint-Luc, Universite´ catholique de Louvain, B-1200 Brussels, Belgium c Department of Pathology, Cliniques universitaires Saint-Luc, Universite´ catholique de Louvain, B-1200 Brussels, Belgium Received 8 November 2006; accepted 15 January 2007
Abstract Deoxycytidine kinase (dCK) activates several antileukaemic nucleoside analogues. We have recently reported that the activity of dCK, overexpressed in HEK 293T cells, correlates with its phosphorylation level on Ser-74. Here, we show that dCK from B-cell chronic lymphocytic leukaemia (B-CLL) lymphocytes can be detected by an anti-phospho-Ser-74 antibody and that interindividual variability in dCK activity is related to its phosphorylation level on Ser-74. Moreover, pharmacological intervention modified Ser-74 phosphorylation, in close parallel with changes in dCK activity. These results suggest that activation of dCK via phosphorylation of Ser-74 might constitute a new therapeutic strategy to enhance activation and efficacy of nucleoside analogues. 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: B-CLL lymphocytes; Deoxycytidine kinase; Immunocytochemistry; Nucleoside analogues; Ser-74 phosphorylation
1. Introduction Phosphorylation of chemotherapeutically important nucleoside analogues, such as fludarabine
*
Corresponding author. Address: Department of Hematology, Cliniques universitaires Saint-Luc, Universite´ catholique de Louvain, B-1200 Brussels, Belgium. Tel.: +32 27647568; fax: +32 27647598. E-mail address: [email protected] (E. Van Den Neste).
(9-b-D-arabinosyl-2-fluoroadenine) or CdA (2-chloro-2 0 -deoxyadenosine), by deoxycytidine kinase (dCK; EC 2.7.1.74) is a prerequisite for their pharmacological action [1]. Identification of mechanisms controlling dCK activity is of particular interest because down-regulation of this enzyme can decrease chemosensitivity to nucleoside analogues in diseases such as B-cell chronic lymphocytic leukaemia (B-CLL) [2–4]. We, and others, have shown that agents known to modify the phosphorylation status of proteins can modulate dCK activity
0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.01.013
C. Smal et al. / Cancer Letters 253 (2007) 68–73
in various types of leukaemic cells, including B-CLL lymphocytes [5–7]. Recently, we have identified by mass spectrometry multiple phosphorylation sites in dCK after overexpression in HEK 293T cells, the most abundant of these being Ser-74. Site-directed mutagenesis demonstrated that Ser-74 phosphorylation was crucial for dCK activity in the latter model [8]. The purpose of the present study was to explore phosphorylation of dCK on Ser-74 in lymphocytes from patients with B-CLL, and its relation to dCK activity. 2. Materials and methods 2.1. Materials Ficoll–Paque Plus (density: 1.077), [5-3H]-deoxycytidine (21 Ci/mmol), Hybond C-extra membranes, ECL enhanced chemiluminescence kit were from GE Healthcare. FCS and penicillin–streptomycin were purchased from BioWhittaker Europe. RPMI-1640 and all tissue culture reagents were from Gibco/Invitrogen. Anti-dCK antibody raised against the C-terminal peptide of human dCK (amino acids 246–260) was generated by Eurogentec, according to the procedure of Hatzis et al. [9]. Anti-phospho-Ser-74 antibody was obtained as described in [8]. Horseradish peroxidase conjugated anti-rabbit antibody was purchased from Sigma–Aldrich. CdA was synthesised and supplied by Prof. J. Marchand (Laboratory of Organic Chemistry, Universite´ catholique de Louvain, Louvain-la-Neuve). Stock solutions of CdA were prepared in ethanol/150 mM NaCl (v/v). Other chemicals, materials and reagents were from Sigma, Merck Biosciences or Bio-Rad Laboratories. 2.2. Cell isolation and incubation Freshly obtained peripheral blood from B-CLL patients was fractionated by Ficoll–Paque sedimentation. Mononuclear cells (P95% B-CLL cells, as confirmed by flow cytometry analysis) were washed and directly lysed, or resuspended in RPMI-1640, supplemented with 10% FCS and 1% penicillin–streptomycin, and incubated at 37 C in 5% CO2 in air. All patients had an established diagnosis of B-CLL, were free of any anticancer treatment for at least 3 months, had lymphocytes >30 · 109/L, and had given informed consent according to institutional guidelines [10]. 2.3. dCK assay and immunoblotting Cell lysis and dCK assay with 10 lM deoxycytidine and 5 mM MgATP as substrates were performed as described in [5]. Immunoblotting with anti-dCK antibody or anti-phospho-Ser-74 antibody was carried out as
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reported in [8]. dCK expression or phosphorylation were quantified by using National Institutes of Health image software. 2.4. Immunocytochemistry The cells (105 cells/cytospin) were cytocentrifuged and fixed in 10% formol for 10 min. Endogenous peroxidases were blocked (peroxidase blocking kit-DAKO), and non-specific antibody staining was prevented by preincubation in 10% normal goat serum for 30 min. Cytospins were then incubated with the phosphospecific antibody (diluted 1/5), after purification by affinity chromatography on phosphopeptide-coupled thiol Sepharose resin (GE Healthcare), as previously described [11]. They were revealed using the Envision system (DAKO) with diaminobenzidine (Sigma–Aldrich). The samples were counterstained using hematoxylin, and examined under light microscopy. 2.5. Statistical analysis The statistical analysis was performed using the twotailed Student’s t-test and the Pearson correlation coefficient. Statistical significance was considered when P values were less than 0.05. 3. Results and discussion We first sought to investigate whether dCK was phosphorylated on Ser-74 in B-CLL cells by probing cell lysates with the anti-phospho-Ser-74 antibody. Our previous work had demonstrated that this antibody is specific for a phosphoresidue in that it fails to detect overexpressed dCK in HEK 293T cell lysates treated with k-protein phosphatase [8]. As illustrated in Fig. 1a (upper panel), a signal is detected with the anti-phospho-Ser-74 antibody at the dCK level. As it is suppressed in the presence of an excess of the phosphopeptide used to generate the antibody (not illustrated), we can conclude that it really corresponds to a phosphorylation of Ser-74. These results accord with our findings in CCRF-CEM cells [8]. Interestingly, the level of phosphorylation of dCK on Ser-74 was variable amongst patients, ranging from almost undetectable (lanes 1, 5, 6, 9) to strong signals (lanes 2, 3, 7, 8). Probing with an anti-dCK antibody (Fig. 1a, lower panel) showed that the amount of dCK protein also varied amongst patients, and was not strictly correlated with the level of phosphorylation of dCK. Indeed, patients with relatively similar levels of total dCK (i.e., lanes 2, 3, 4, 6, 8) displayed considerably different states of Ser-74 phosphorylation. As given in Fig. 1a, values of dCK activity also displayed broad variations between patients. After quantification of dCK phosphorylation and dCK level by densitometric analysis in samples of 15 different patients, dCK activity was plotted against phospho-dCK/total dCK ratio (Fig. 1b) or
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pSer-74-dCK (arbitrary units) Fig. 1. dCK phosphorylation on Ser-74 in B-CLL lymphocytes and its relation to dCK activity. (a) Lysates (100 lg) of lymphocytes from ten B-CLL patients were subjected to SDS–PAGE followed by immunoblotting with the anti-phospho-Ser-74 antibody (upper part), or with the anti-dCK antibody as a control for dCK expression (lower part). dCK activity, expressed in pmol/min/mg of protein, is given below each corresponding patient. (b) dCK activity was plotted against phospho-dCK/total dCK ratio, assessed by densitometric quantification, in 15 B-CLL samples (including in addition the five control samples of Fig. 2). Analysis by linear least squares regression gave r = 0.90 with P < 0.0001. (c) dCK activity was plotted against phospho-dCK and analyzed as in b (r = 0.95; P < 0.0001).
against phospho-dCK alone (Fig. 1c). Analysis by linear least squares regression showed a significant positive correlation between dCK activity and both phospho-dCK/total dCK ratio (r = 0.90) or phospho-dCK (r = 0.95), indicating that the phosphorylation state of dCK plays
an important role in the level of basal dCK activity in BCLL cells. It is interesting to note that the correlation between dCK activity and dCK phosphorylation is better than the correlation previously established between dCK activity and dCK content (r = 0.554) [12].
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Our next objective was to explore whether the dCK Ser-74 phosphorylation level could be modified in BCLL cells, and whether this modification would correlate with changes in dCK activity. For this purpose, B-CLL cells were treated with various agents able to increase or decrease dCK activity [5–8,13,14]. As shown in Fig. 2 (patients 11–13 and 15), CdA, etoposide (VP-16), UV-C irradiation, genistein (GNT) and aphidicolin (APC) all increased dCK phosphorylation on Ser-74, in close parallel with dCK activity. These results strongly suggest that all the agents tested increase dCK activity via phosphorylation of Ser-74. Conversely, hyperosmotic stress induced by sorbitol (SORB) significantly reduced Ser-74 phosphorylation, as well as dCK activity in BCLL lymphocytes (patient 15), as previously observed in dCK-overexpressing HEK 293T cells [8]. Fig. 2 also illustrates a sample (patient 14) in which SORB did not significantly reduce dCK activity, probably because basal phosphorylation of dCK was already at a very low level. In each patient, expression of total dCK remained unchanged in all conditions tested. Taken together, our results validate the previous hypothesis that dCK activity is regulated by reversible phosphorylation in leukaemic cells [5,6,8,13,14], although other mechanisms besides phosphorylation might also be involved in the control of this enzyme activity [15]. Indeed, in patient 12, there is clearly a close parallellism between increase in Ser-74 phosphorylation and dCK activity, but the values of dCK activity after treatment are lower than in patients 11 and 13, despite an apparently higher ratio phosphodCK/total dCK.
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Finally, we performed immunocytochemistry with purified anti-phospho-Ser-74 antibody in both CCRFCEM and B-CLL cells to analyze whether increase of Ser-74 phosphorylation could be detected in situ and could modify subcellular localization of phospho-dCK (Fig. 3). No staining of CCRF-CEM cells was detected in the absence of the primary anti-phospho-antibody (Fig. 3a). In contrast, cytoplasmic staining was seen with the anti-phospho-Ser-74 antibody (Fig. 3b), confirming recent results with an antibody directed against the C-terminal end of the dCK protein [16]. Moreover, staining was increased after incubation with VP-16 (Fig. 3c), which activates dCK in this cell line [13]. The specificity of the staining was ensured by adding an excess of the phosphopeptide used to raise anti-Ser-74 antibodies in rabbit, which almost completely abolished immunostaining (Fig. 3d). Phospho-dCK was also located in the cytoplasm of most of control B-CLL lymphocytes, with a tendency to scatter in perinuclear areas (Fig. 3e). After incubation with CdA (Fig. 1f), VP-16 (Fig. 3g), or after UV irradiation (Fig. 3h), most B-CLL lymphocytes examined were also positive for phospho-Ser-74 dCK, but with a greater intensity of staining with respect to control cells. The immunostaining of treated cells had a granular appearance mainly expressed in perinuclear and submembrane regions, which is not in favor of a translocation of dCK from the cytoplasm into the nucleus when it is activated by phosphorylation. In the same samples, we confirmed that dCK activity (see insets of Fig. 3b–h) and dCK phosphorylation on Ser-74 (not shown) were increased by CdA, VP-16 or UV-light.
Fig. 2. Phosphorylation of dCK on Ser-74 in relation with changes in dCK activity. B-CLL lymphocytes were incubated without () or with 1 lM CdA, 100 lM etoposide (VP-16), 100 lM genistein (GNT) or 10 lM aphidicolin (APC) for 2 h, or with 0.5 M sorbitol (SORB) for 1 h, or UV-C-radiated (30 J/m2) and thereafter incubated for 30 min (or 2 h for patient 13). Cell lysates (50–100 lg) were analysed for dCK phosphorylation on Ser-74 and dCK content as in Fig. 1a, and for dCK activity.
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Fig. 3. Immunocytochemical staining of dCK phosphorylated on Ser-74 in CCRF-CEM (a–d) or in B-CLL lymphocytes (e–h). Cytospins were prepared and stained with anti-phospho-Ser-74 antibody as described in Section 2. (a) Negative control. (b) Untreated CCRF-CEM cells. (c) CCRF-CEM cells treated with 100 lM VP-16 for 2 h. (d) Same as in (c) except that the anti-phospho-Ser-74 antibody was blocked with the antigenic phosphopeptide. (e) Untreated B-CLL cells. (f) B-CLL cells treated with 1 lM CdA for 2 h. (g) B-CLL cells treated with 100 lM VP-16 for 2 h. (h) B-CLL cells incubated for 30 min after UV-C radiation (30 J/m2). dCK activity (pmol/min/mg protein) was determined in parallel samples (insets). Images were acquired using a Zeiss Axioplan microscope (Zeiss, Go¨ttingen, Germany) with a 40· Plan Neofluar objective (NA 0.75). Images were captured with a HRc CDD camera (Axiocam, Zeiss), and processed using Axiovision software (Zeiss).
We conclude that phosphorylation of dCK on Ser-74 is crucial for dCK activity in B-CLL cells. This accords with our previous finding that mutation of Ser-74 to a nonphosphorylatable residue strongly decreased human dCK activity overexpressed in HEK 293T cells [8]. This post-translational mechanism of regulation is likely to be implicated in the high variability of dCK activity amongst B-CLL patients, which is not satisfactorily explained by changes in dCK mRNA levels [17]. It should be noted, however, that other mechanisms of dCK inactivation have been reported in patients with acute myeloblastic leukaemia (AML), including alternatively spliced dCK mRNA transcripts [18], or single nucleotide polymorphisms in the promoter region of dCK [19]. Mutation in the dCK gene rarely occurs in AML patients [20–22]. Finally, because phosphorylation of dCK on Ser-74 can be increased by pharmacological intervention, our results may reinforce strategies aimed at restoring or increasing dCK activity to improve clinical efficacy of nucleoside analogues in B-CLL [23,24]. Indeed, uptake of nucleoside analogues, which includes transport through the plasma membrane, but also their phosphorylation, may be reduced in some patients, causing nucleoside analogue resistance [3]. Studies are in progress to assess the relationship between phosphorylation of dCK on Ser-74, activation of various nucleoside analogues, and in vitro sensitivity to the latter drugs.
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