Tatjana Spasokoukotskaja

Expression of deoxycytidine kinase and phosphorylation of 2-chlorodeoxyadenosine in human normal and tumour cells and tissues.

Deoxycytidine kinase (dCK) activates several clinically important drugs, including the recently developed antileukaemic compound 2-chlorodeoxyadenosine (CdA). The distribution of dCK in cells and tissues has previously been determined by activity measurements, which may be unreliable because of the presence of other enzymes with overlapping substrate specificities. Therefore we have measured dCK polypeptide levels in extracts of normal and malignant human peripheral blood mononuclear cells, gastrointestinal tissues and sarcomas, using a specific immunoblotting technique, as well as the phosphorylation of CdA in the same extracts. High levels of dCK were found in all major subpopulations of normal mononuclear leucocytes (120 +/- 19 ng dCK/mg protein) and in B-cell chronic lymphocytic leukaemia (81 +/- 30 ng/mg, n = 23). Hairy-cell leukaemia contained lower levels (28 +/- 23 ng/mg, n = 7), as did three samples of T-cell chronic lymphocytic leukaemia (18 +/- 14 ng/mg). Phytohaemagglutinin stimulation of normal lymphocytes did not lead to any substantial increase in either dCK activity or protein expression (less than 2.5-fold). The human CEM wt T-lymphoblastoid cell line contained 56 +/- 1 ng/dCK/mg protein, while in the CEM ddC50 and AraC8D mutants that lack dCK activity, no dCK polypeptide could be detected. In colon adenocarcinomas, the dCK content was significantly higher (20 +/- 9 ng/mg, n = 20) than in normal colon mucosa (8 +/- 3.5 ng/mg, n = 19, P < 0.05). A similar pattern of dCK expression was found in gastric adenocarcinomas (21 +/- 13 ng/mg, n = 5) and normal stomach mucosa (6 +/- 5 ng/mg, n = 5, P < 0.15). One leiomyosarcoma and one extra-skeletal osteosarcoma showed dCK levels comparable with those found in normal lymphocytes (84 +/- 6 and 109 +/- 4 ng/mg, respectively), while other sarcoma samples contained lower levels, comparable to the gastrointestinal adenocarcinomas (20 +/- 7 ng/mg, n = 12). Thus, dCK is expressed constitutively and predominantly in lymphoid cells, but it is also found in solid non-lymphoid tissues, with increased levels in malignant cells. The phosphorylation of CdA in crude extracts showed a close correlation to the dCK polypeptide level.

Properties and levels of deoxynucleoside kinases in normal and tumor cells; implications for chemotherapy

Deoxynucleoside kinases are key enzymes in deoxyribonucleoside salvage, activating several clinically important chemotherapeutic drugs. The four known kinases, cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK) and the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK), have been purified and characterized as to the subunit structure as well as specificity with a large number of analogs. These results are summarized and used to establish selective assays for the four enzymes in crude extracts of normal and malignant human peripheral blood mononuclear cells, gastrointestinal tissues and sarcomas. TK2 and dGK activities were found at low levels in all tissues, possibly correlated to the content of mitochondria. TK1 activity was detected only in samples containing a significant number of S phase cells. We have measured dCK activity as well as dCK polypeptide level by immuno blotting in these extracts. High levels of dCK were found in normal mononuclear leukocytes (91-145 ng dCK/mg protein) and in B-cell chronic lymphocytic leukemia (80 +/- 30 ng/mg, n = 23). Hairy cell leukemia contained lower levels (28 +/- 23 ng/mg, n = 7), as did unexpectedly three samples of T-cell chronic lymphocytic leukemia (18 +/- 14 ng/mg). Phytohemaglutinine stimulation of normal lymphocytes did not lead to any substantial increase in either dCK activity or expression (less than 2.5-fold). In colon adenocarcinomas, the dCK content was significantly higher (21 +/- 9.3 ng/mg, n = 20) than in normal colon mucosa (8.2 +/- 3.7 ng/mg, n = 19, p < 0.05). A similar pattern of dCK expression was found in gastric adenocarcinomas (21 +/- 13 ng/mg, n = 5) and normal ventricular mucosa (6.2 +/- 5.4 ng/mg, n = 5, p < 0.15). One leiomyosarcoma and one extra-skeletal osteosarcoma showed a dCK levels comparable to those found in normal lymphocytes (84 +/- 6 and 109 +/- 4 ng/mg), while other sarcoma samples contained levels comparable to the gastrointestinal adenocarcinomas (20 +/- 7 ng/mg, n = 12). We confirm that dCK is expressed constitutively and predominantly in lymphoid cells, but conclude that a significant expression may be found in non-lymphoid tissues as well, with increased levels in the corresponding tumor tissue. 2-Chlorodeoxyadenosine (CdA), an antileukemic agent used in treatment of hairy cell leukemia and chronic lymphocytic leukemias (B-CLL), is phosphorylated by dCK which was used as the selective substrate for this enzyme. A study was performed to investigate if there was a correlation between the dCK levels and the response to CdA treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of deoxycytidine kinase by inhibition of DNA synthesis in human lymphocytes

Deoxycytidine kinase (dCK, EC.2.7.1.74) is a key enzyme in the intracellular metabolism of 2-chlorodeoxyadenosine, 1-beta-D-arabinofuranosylcytosine, difluorodeoxycytidine, and other drugs used in chemotherapy of different leukaemias and solid tumours. Recently, stimulation of dCK activity was shown by these analogues and by other genotoxic agents such as etoposide and NaF, all of which cause severe inhibition of DNA synthesis in cell cultures. Here we describe that direct inhibition of DNA polymerases by aphidicolin stimulated dCK activity in normal lymphocytes and acute myeloid leukaemic cells, as well as in HL 60 promyelocytic cell cultures. Increased dCK activity was not due to new protein synthesis under our conditions, as measured by immunoblotting. Partial purification by diethylaminoethyl-Sephadex chromatography revealed that the activated form of dCK survived purification procedure. Moreover, it was possible to inactivate purified dCK preparations by recombinant protein phosphatase with Ser/Thr/Tyr dephosphorylating activity. These data suggest that the activation of dCK may be due to phosphorylation, and that deoxynucleoside salvage is promoted during inhibition of DNA synthesis in human lymphocytes.

Activation of deoxycytidine kinase by gamma-irradiation and inactivation by hyperosmotic shock in human lymphocytes

Deoxycytidine kinase (dCK) is a key enzyme in the intracellular metabolism of deoxynucleosides and their analogues, phosphorylating a wide range of drugs used in the chemotherapy of leukaemia and solid tumours. Previously, we found that activity of dCK can be enhanced by incubating primary cultures of lymphocytes with substrate analogues of the enzyme, as well as with various genotoxic agents. Here we present evidence that exposure of human lymphocytes to 0.5-2 Gy dosage of gamma-radiation as well as incubation of cells with calyculin A, a potent inhibitor of protein phosphatase 1 and 2A, both elevate dCK activity without changing the level of dCK protein. When cells were gamma-irradiated in the presence of calyculin A, a more pronounced activation of dCK was observed. In contrast, both basal and stimulated dCK activities were reduced by hyperosmotic treatment of the cells. DNA repair determined by the Comet assay and by thymidine incorporation was induced by irradiation. Complete repair of gamma-irradiated DNA was detected within 1 hr following the irradiation along with dCK activation, but the rate of repair was not accelerated by calyculin A. These data provide evidence for the activation of dCK upon DNA damage and repair that seems to be mediated by phosphorylation of the enzyme, suggesting the role of dCK in DNA repair processes.

Phosphorylation of 2-chlorodeoxyadenosine (CdA) in extracts of peripheral blood mononuclear cells of leukaemic patients

2‐Chlorodeoxyadenosine (CdA) is an antileukaemic agent used in treatment of hairy cell leukaemia (HCL) and chronic lymphocytic leukaemia of B‐ and T‐cell type (B‐CLL and T‐CLL). The aim of this study was to elucidate the interpatient variability of CdA phosphorylation and its relation to response to CdA treatment. In extracts of peripheral blood mononuclear cells of patients with B‐CLL (n= 39), CdA phosphorylation was significantly higher than in HCL (n= 19) when calculated per protein (391 ± 155 pmol CdA phosphorylated/mg protein/min versus 288 ± 166 pmol/mg/min, P < 0.001), but was the same when calculated per cell (12 ± 5.9 pmol/106 cells/min versus 14 ± 5.9 pmol/106 cells/min) due to a larger cell volume in HCL. In T‐CLL (n= 6), CdA phosphorylation was significantly lower than in B‐CLL, both when calculated per protein (128 ± 68 pmol/mg/min, P < 0.001) or per cell (5.7 ± 2.7 pmol/106 cells/min, P < 0.05). This low CdA phosphorylation in T‐CLL was unexpected because normal B‐ and T‐lymphocytes contain equal amounts of CdA phosphorylation. With B‐CLL, 21 patients who responded (complete and partial response) to CdA treatment showed a significantly higher CdA phosphorylation than 13 patients not responding to CdA treatment (456 ± 170 pmol/mg/min versus 309 ± 97 pmol/mg/min, P < 0.01). We conclude that the level of CdA phosphorylation is correlated with the response of leukaemias to CdA treatment.

Bcl-2 rearrangements with breakpoints in both vcr and mbr in non-Hodgkin’s lymphomas and chronic lymphocytic leukaemia

The bcl‐2 gene is rearranged in most cases of follicular lymphoma and the breakpoint clusters into two specific regions: mbr and mcr. Rearrangements to immunoglobulin heavy chain genes (IgH) result in a deregulation of the gene and increased transcription of mRNA for the bcl‐2 protein. In chronic lymphocytic leukaemia (CLL) expression of bcl‐2 protein is increased but rearrangement of the gene can be found only in a minority of cases; commonly a variant translocation with a breakpoint region located 5′ of the bcl‐2 gene (vcr) with preferential rearrangement to immunoglobulin light chain genes. We have analysed breakpoints in mbr and vcr in malignant cells from 96 patients with B‐CLL, 45 with hairy cell leukaemia (HCL) and 41 with high‐ and low‐grade non‐Hodgkin’s lymphomas (NHL). Vcr rearrangements were observed in nine patients (12%) with B‐CLL. Four patients had co‐migration of rearranged bcl‐2 bands to kappa genes and two patients to IgH. Cytogenetic abnormalities involving 18q21, the site of the bcl‐2 gene, was found in two cases only. In several cases with bcl‐2 gene rearrangement chromosomal aberrations not including 18q21 were observed. In six patients (two B‐CLL, one follicular lymphoma, one immunocytoma and two high‐grade lymphomas), breakpoints in both vcr and mbr were found. In HCL a rearrangement in the vcr region was found in one case. Bcl‐2 protein immunostaining of B‐CLL showed intense bcl‐2 expression in all cases and no correlation was found between gene rearrangement and protein expression. Our study confirms that breakpoints in the bcl‐2 gene commonly cluster to the vcr region in B‐CLL, but in most cases over‐expression of bcl‐2 protein has to be explained by other mechanisms than bcl‐2 gene rearrangement. We also report that simultaneous breakpoints in mbr and vcr is a recurrent phenomenon in B‐CLL and in other high‐ and low‐grade non‐Hodgkin’s lymphomas.

Activation of Deoxycytidine Kinase by Various Nucleoside Analogues

The effect of different nucleoside analogues on deoxycytidine kinase (dCK) and thymidine kinase (TK) was compared in normal human lymphocytes and various leukemic cell lines. G-phase enriched tonsilar lymphocyte subpopulation treated by CdA showed more profound stimulation of dCK activity than S-phase cells. No substantial changes in TK activity were detected. CdA treatment increased the activity of dCK 4-fold in peripheral blood mononuclear cells (PBMC) and 2-fold in promyelocytic cell line HL60, too. However, no significant stimulation was detected either in CCRF-CEM or in K562 cell lines. 2-Cl-2′deoxy-2′F-adenine arabinoside (CAFdA), 2F-adenine arabinoside (F-araA) and cytosine arabinoside (AraC) had the same effect as CdA, although higher concentrations were needed for maximal activation. In contrast, treatment by dCyd caused slight inhibition of dCK. The possibility of interference of nucleoside analogues with the mechanisms of posttranslational modification of dCK was proposed.

EFFECT OF PHOSPHORYLATION ON DEOXYCYTIDINE KINASE ACTIVITY

In contrast to deoxycytidine kinase from leukemic blasts7, the recombinant dCK is a poor substrate for protein kinase C. Protein kinase A effectively phosphorylates the recombinant dCK. However, neither substrate specificity, nor kinetic parameters of dCK were changed upon phosphorylation. Nevertheless, treatment of partially purified lymphocyte dCK with recombinant phosphoprotein phosphatase completely destroys its phosphotransferase activity. These data suggest that phosphorylation plays a role in the regulation of lymphocyte dCK

Deoxycytidine Kinase is Reversibly Phosphorylated in Normal Human Lymphocytes

The activity of deoxycytidine kinase (dCK) has been shown to be enhanced upon genotoxic stress in human lymphocytes, and reversible phosphorylation of the enzyme has been implicated in the activation process. Here, we provide compelling evidence that dCK is a cytosolic phosphoprotein. Two-dimensional gel electrophoresis revealed that dCK has several differentially charged isoforms in cells. One-third of total cellular dCK was bound to a phosphoprotein-binding column irrespective of its activity levels, indicating that other mechanisms rather than phosphorylation alone might also be involved in the stimulation of enzyme activity. We excluded the possibility that activated dCK is translocated to the nucleus, but identified a dCK isoform of low abundance with a higher molecular weight in the nuclear fractions.

Selective Increase of dATP Pools upon Activation of Deoxycytidine Kinase in Lymphocytes: Implications in Apoptosis

Stimulation of the activity of deoxycytidine kinase (dCK), the principal deoxynucleoside salvage enzyme, has been recently considered as a protective cellular response to a wide range of agents interfering with DNA repair and apoptosis. In light of this, the potential contribution of dCK activation to apoptosis induction—presumably by supplying dATP or its analogues for the apoptosome formation—deserves consideration. Two‐hour exposure of human tonsillar lymphocytes to 2‐chloro‐deoxyadenosine (CdA) led to a two‐fold activation of dCK. This activation process was inhibited by pifithrin‐α, a potent inhibitor of p53. When the dNTP pools were determined, both deoxypyrimidine triphosphate and dGTP pools were reduced after the treatments, while dATP levels elevated by 62%, 77% and 50% in the CdA, aphidicolin and etoposide‐treated cells, respectively. We assume that dCK activation elicited by cellular damage might be a proapoptotic factor in terms of generating dATP well before the release of cytochrome c and deoxyguanosine kinase from mitochondria.