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

doi:10.1016/0959-8049(94)00435-8

European Journal of Cancer

Volume 31, Issue 2, 1995, Pages 202-208

https://doi.org/10.1016/0959-8049(94)00435-8 Get rights and content

Abstract

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, a = 12). Thus, dCK is expressed constitutively and predominantly in lymphoid cells, but it is also found in solid non-Iymphoid tissues, with increased levels in malignant cells. The phosphorylation of CdA in crude extracts showed a close correlation to the dCK polypeptide level.

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Antibody-based methods such as western blotting and enzyme-linked immunosorbent assay have limited ability to detect the active form of dCK; therefore, antibodies that can recognize various dCK substrates are required [13,14]. Conventional assays for dCK, based on the use of radioactive substrates such as [8-3H] CdA and a series of binding and washing procedures to separate “hot” and “cold” portions to assess activity [4,14], are complicated with the use of radioisotope materials, which involves problems such as waste disposal and radiation safety. Bierau et al [15] developed a high-performance liquid chromatography (HPLC) method using nonradiolabeled CdA with inherent limitation in sensitivity because nucleosides have no specific chromophores for spectrophotometric detection.
A new analytical method for determining deoxycytidine kinase (dCK) activity in biological milieus using luminescence is reported here. This method, based on utilizing adenosine triphosphate (ATP) as the sole phosphate donor in the kinase reaction and monitoring ATP consumption via a luciferase-based chemiluminescence reaction, is capable of detecting dCK activity without the use of specific substrates or radioisotope techniques. Comparing with the reverse-phase high-performance liquid chromatography method, this new method is suggested to be efficient and sensitive. Further, application of the proposed method for profiling dCK activity in cultured cancer cells revealed that a cervix cell line exhibited the highest dCK activity to gemcitabine metabolism.
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In lymphomas, as in other tumor types, 2 major factors determine the clinical activity of gemcitabine and other nucleoside analogs: substrate specificity for their activating nucleoside kinases and the expression of these enzymes in tumor tissues. The content of dCK, the rate-limiting enzyme in the activation of pyrimidine analogs, is severalfold higher in lymphocytes than in epithelial cells, and in most tumor tissues compared to their normal counterparts [35,36]. The affinity of dCK for gemcitabine is higher than for fludarabine, cytarabine, or cladribine, which may account for its broader spectrum of activity [12].
We developed a new high-dose combination of infusional gemcitabine with busulfan and melphalan for lymphoid tumors. Gemcitabine dose was escalated by extending infusions at a fixed rate of 10 mg/m²/min in sequential cohorts, in daily, 3-dose or 2-dose schedules. Each gemcitabine dose immediately preceded busulfan (adjusted targeting area under the curve 4,000 μM/min⁻¹/day × 4 days) or melphalan (60 mg/m²/day × 2 days). We enrolled 133 patients (80 Hodgkin lymphoma [HL], 46 non-Hodgkin lymphoma [NHL], 7 myeloma), median 3 prior regimens; primary refractory disease in 63% HL/45% NHL and positron emission tomography positive tumors at transplantation in 50% patients. Two patients died from early posttransplantation infections. The major toxicity was mucositis. The daily and 3-dose schedules caused substantial cutaneous toxicity. In contrast, the 2-dose schedule was better tolerated, which allowed us to extend the infusions from 15 to 270 minutes. Pretransplantation values of C-reactive protein, B-type natriuretic peptide, ferritin, or haptoglobin did not correlate with toxicity. Overall response and complete response rates were 87%/62% (HL), 100%/69% B large-cell lymphoma (B-LCL), 66%/66% (T-NHL), and 71%/57% (myeloma). At median follow-up of 24 months (range, 3-63 months), the event-free/overall survival rates were 54%/72% (HL), 60%/89% (B-LCL), 70%/70% (T-NHL), and 43%/43% (myeloma). In conclusion, gemcitabine/busulfan/melphalan is a feasible regimen with substantial activity against a range of lymphoid malignancies. This regimen merits further evaluation in phase II and III trials.
Relationship between deoxycytidine kinase (DCK) genotypic variants and fludarabine toxicity in patients with follicular lymphoma
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DCK catalyzes the intracellular phosphorylation of fludarabine. The promoter and coding region of the DCK gene were analyzed in 74 follicular lymphoma (FL) patients receiving a therapeutic regimen that included fludarabine. DCK mRNA expression was quantified in a cohort of healthy donors. Four previously described genotypic variants, −360C>G, −201C>T (rs2306744), C28624T (rs11544786) and c.91+37G>C (rs9997790), as well as the new variant, −12C>G, were identified. Variant C28624T showed a lower risk of lymphopenia (P = 0.04), but a higher risk of neutropenia (P = 0.04). Statistical significance was found in bivariate logistic regression between lymphopenia and infectious episodes in the induction period (odds ratio 3.85, P = 0.04).
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These kinases are implicated in a variety of cellular functions, including vesicle trafficking, DNA repair, cell division and progression, circadian rhythms and apoptosis [17]. Interestingly, CKI δ, like dCK, has been shown to display high activity in lymphocytes [18,19] and to play an important role in the DNA damage response [9,20,21]. Moreover, CKI δ protein levels were found to be p53-dependently elevated upon etoposide treatment [20] that also induces increase of Ser-74 phosphorylation and dCK activity in leukemic cells [7,14].
Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI δ. We showed that recombinant CKI δ phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI δ correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI δ, strengthening the key role of this residue in the control of dCK activity. However, neither CKI δ inhibitors nor CKI δ siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI δ in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI δ could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure–activity relationships in the enzyme.
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Thus, dCK and dCDA are two important enzymes in the activation and inactivation of gemcitabine, respectively. Increased dCK levels have been observed in human cells of various malignancies.13,14 This could give an advantage for liver malignancies compared to normal liver tissue in selectively activating the gemcitabine delivered by HAI.
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^☆: Parts of the results have been presented in the proceedings of a scientific meeting: Eriksson S, Arnér E, Spasokoukotskaja T, et al. Properties and levels of deoxynucleoside kinases in normal and tumor cells; implications for chemotherapy. Advan. Enzyme Regul. 1994, 34, 13-25.

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PaperExpression of deoxycytidine kinase and phosphorylation of 2-chlorodeoxyadenosine in human normal and tumour cells and tissues☆

Abstract

J Biol Chem

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

J Biol Chem

Biochem Biophys Res Commun

Pharmacol Ther

EurJ Cancer

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

Eur J Cancer Clin Oncol

Exp Cell Res

Leuk Res

Exp Cell Res

Biochem Pharmacol

Biochem Biophys Res Commun

J Biol Chem

Lancet

Biochem Pharmacol

Cell Immunol

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