Saturation of intracellular cytosine arabinoside triphosphate accumulation in human leukemic blast cells

doi:10.1016/0145-2126(90)90035-8

Leukemia Research

Volume 14, Issue 5, 1990, Pages 475-479

https://doi.org/10.1016/0145-2126(90)90035-8 Get rights and content

Abstract

Accumulation of cytosine arabinoside triphosphate (araCTP) from a range of cytosine arabinoside (araC) concentrations (1–50 μM) was measured during incubations of leukemic cells freshly isolated from patients with acute leukemia. In all but one patient, increments in extracellular araC above 10 μM did not increase intracellular araCTP levels. This maximal level of araCTP accumulation ranged from 254 to 1607 pmol/10⁷ cells attained after 1 h incubation and did not correlate with either the number of nucleoside transporters on the cell membrane or the V_max of araC phosphorylation in cell free extracts. Extremely low araCTP accumulation (103 pmol/10⁷ cells/h at 50 μM araC) was observed in an AML patient with the unusual finding of micromyeloblasts. These cells also had very low numbers of nucleoside transport sites (< 500 sites/cell) and were mitotically inactive. The unique feature of the myeloblasts from this patient was that intracellular araCTP accumulation showed a linear dependence on extracellular araC up to 50 μM with no evidence of saturation.

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Saturation of 1-β-d-arabinofuranosylcytosine 5′-triphosphate accumulation in leukemia cells during high-dose 1-β-d-arabinofuranosylcytosine therapy

Cancer Res.

(1987)

Cited by (29)

Expression of high Km 5′-nucleotidase in leukemic blasts is an independent prognostic factor in adults with acute myeloid leukemia
2001, Blood
Citation Excerpt :
Ara-C catabolism can result from rapid deamination by cytidine deaminase (CDD) to the nontoxic metabolite ara-U.16 Cytoplasmic 5′-nucleotidase (5NT) activities oppose that of dCK by dephosphorylating ara-CMP, thereby preventing the production of the active form.17,18 In vitro data suggest that modifications of ara-C transport and metabolism processes affect intracellular ara-CTP concentration, inducing drug resistance.19–21 Deficiency of hENT1, dCK, and overexpression of CDD or high Km 5NT have all been reported to be associated with resistance to ara-C in different cell lines.22–25
Cytarabine (ara-C) requires activation into its triphosphorylated form, ara-CTP, to exert cytotoxic activity. Cytoplasmic 5′-nucleotidase (5NT) dephosphorylates ara-CMP, a key intermediate, preventing accumulation of ara-CTP and may reduce cellular sensitivity to the cytotoxic activity of ara-C. To determine whether the level of expression of 5NT is correlated with clinical outcome in patients with acute myeloid leukemia (AML) treated with ara-C, this study analyzed the levels of messenger RNA expression of high Km 5NT by real-time polymerase chain reaction at diagnosis in blast cells of 108 patients with AML. High Km 5NT was expressed at diagnosis in the blast cells of 54% of patients. In univariate analysis, (1) patients whose blast cells contained high levels (values greater than the median value for total population) of high Km 5NT at diagnosis had significantly shorter disease-free survival (DFS) than patients with low levels of high Km 5NT (11 months versus 17.5 months, P = .02) and (2) high levels of high Km 5NT also predicted significantly shorter overall survival (15.7 months versus 39 months, P = .01) in young patients (≤ 57 years; median value for the entire population). In a multivariate analysis taking into account age, karyotype risk, and other factors found to have prognostic significance in univariate analysis, (1) high Km 5NT expression was an independent prognostic factor for DFS and (2) high levels of high Km 5NT also predicted significantly shorter overall survival in young patients. These results demonstrate that the expression of high levels of high Km 5NT in blast cells is correlated with outcome in patients with AML.
Protection of leukemic cells by deoxycytidine: In vitro measures of protection against cytosine arabinoside
1998, Leukemia Research
Plasma deoxycytidine levels can be very high in leukemia patients. Such levels strongly protected leukemia cell lines against cytosine arabinoside (araC), fludarabine and 2-chlorodeoxyadenosine when using clonogenic survival as the endpoint. This endpoint is not easily used when studying protection in clinical leukemia cell samples. Therefore, we tested other ways to quantify protection based on biochemical measures of viability or drug metabolism. The estimates of the strength of protection based on rates of DNA synthesis, cellular araC uptake and incorporation of araC into DNA were much lower than the estimates using clonogenic survival. The MTT viability assay gave excellent estimates and appears promising for studying protection in primary leukemia cell samples.
Nucleoside transport and its significance for anticancer drug resistance
1998, Drug Resistance Updates
This article discusses the role of nucleoside transport processes in the cytotoxicity of clinically important anticancer nucleosides. This article summarizes recent advances in the molecular biology of nucleoside transport proteins, review the current state of knowledge of the transportability of therapeutically useful anticancer nucleosides, and provide an overview of the role of nucleoside transport deficiency as a mechanism of resistance to nucleoside cytotoxicity are summarized. Several strategies for utilization of nucleoside transport processes to improve the therapeutic index of anticancer therapies, including the use of nucleoside-transport inhibitors to modulate toxicity of both nucleoside and non-nucleoside antimetabolite drugs are also presented.
Deoxycytidine in human plasma: Potential for protecting leukemic cells during chemotherapy
1997, Cancer Letters
Degradation of DNA produces deoxycytidine. Metabolism of deoxycytidine to dCTP inhibits phosphorylation of cytosine arabinoside (araC), fludarabine (FaraA) and 2-chlorodeoxyadenosine (CdA) by deoxycytidine kinase. This study measured plasma deoxycytidine in healthy adults and two leukemia patients and then determined how clinically relevant deoxycytidine levels would affect drug toxicity in human leukemia and lymphoma cells. Deoxycytidine was well below 0.05 μM in ten healthy persons. In the leukemia patients it was <0.05 and 0.44 μM before chemotherapy, rising to 10.3 and 5.5 μM during treatment. A broad range of clinically relevant deoxycytidine levels were high enough to profoundly decrease araC, FaraA and CdA toxicity in MOLT3, CA46 and HL60 leukemia/lymphoma cells and to change dCTP, DNA synthesis and drug incorporation into DNA in a manner consistent with prior mechanistic studies. Varying deoxycytidine levels could be an important factor influencing leukemia therapy.
Effects of fludarabine and gemcitabine on human acute myeloid leukemia cell line HL 60: Direct comparison of cytotoxicity and cellular Ara-C uptake enhancement
1996, Leukemia Research
This study was designed to compare the effects of fludarabine and gemcitabine on cytosine arabinoside (Ara-C) uptake and retention, and their specific cytotoxicity on HL 60 human acute myeloid leukemia cells. The leukemic blasts were exposed to either drug at equimolar concentrations (10 μM) for 3 h and further incubated with Ara-C (5 μM), added immediately (day 0) or after an interval of 24 h in which cells were kept in a drug-free medium (day 1). On day 0, leukemic cells exposed to fludarabine 10 μM had a significant (P < 0.01) increase in Ara-C uptake (297 ± 11 pmol/10⁷ cells) with respect to control cells (not pre-treated: 195 ± 10 pmol/10⁷ cells). After treatment of leukemic cells with fludarabine, cytoplasmic Ara-C peaked after 180 min of exposure, as well as nuclear bound Ara-C. At the same time, a significant decrease in the number of S-phase leukemic cells, consistent with depressed [³H]TdR uptake was observed. Although on day 0 gemcitabine 10 μM did not have potentiating effects on Ara-C uptake, it showed a high degree of intrinsic cytotoxicity as a single agent (clear from cell cycle distribution, [³H]TdR uptake, plating efficiency (PE) data and percentage of apoptotic cells). Cells exposed to gemcitabine, on the other hand, showed on day 1 a significant increase in Ara-C uptake (2.4 × control values in the cytoplasmic and 3 × in the nuclear fractions) and a reduced number of S-phase blasts, [³H]TdR uptake and PEs, as well as an increased apoptotic cell death. Evidently, it is possible to modulate Ara-C uptake by leukemic cells with gemcitabine. Although this effect is similar to that demonstrated with fludarabine, its kinetics and time of efficacy are different and also, because of its intrinsic higher cytotoxicity and lack of important side effects, gemcitabine could be considered a suitable candidate for Ara-C association therapy in acute leukemia.
Formation of cytosine arabinoside-5′-triphosphate in different cultured lymphoblastic leukaemic cells with reference to their drug sensitivity
1995, European Journal of Cancer
The accumulation of intracellular cytosine arabinoside-5′-triphosphate (Ara-CTP) is determined in five lymphoblastic cell lines: Molt 4, H9 and three newly established cell lines from paediatric patients, KFB-1, KFB-2, KFT-1. The cell lines KFB-1 and KFB-2 are B-lymphoblastic (B-ALL), the others are T-lymphoblastic leukaemic cells (T-ALL). The Ara-CTP levels were compared with the sensitivity of the cells to Ara-C. The cells were incubated at different concentrations (100 nM-100 μM) of Ara-C for 4 h or incubated for variable times (30 min–11 h) at 0.1,1 and 10 μM Ara-C to form Ara-CTP. The Ara-CTP-concentrations were measured by high pressure liquid chromatography (HPLC). To determine the sensitivity of the cells to Ara-C, the MIT colorimetricassay was used. The studies indicate that different B- and T-lymphoblastic leukaemia cell lines accumulate Ara-CTP to a markedly different extent. Ara-CTP plateau levels and sensitivity of the cells to Ara-C correlated well in four of the five cells lines studied.

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Leukemia Research

Abstract

References (23)

High-dose cytosine arabinoside therapy for refractory leukemia

Blood

Relationships among ara-CTP pools, formation of (ara-C)DNA, and cytotoxicity of human leukemic cells

Blood

Pharmacokinetics of continuous intravenous and subcutaneous infusions of cytosine arabinoside

Blood

Quantitative extraction and estimation of intracellular nucleoside triphosphates of Escherichia coli

Analyt. Biochem.

High dose cytarabine: a review

Leukemia

Lethality of human myeloblasts correlates with the incorporation of arabinofuranosylcytosine into DNA

Relationship of 1-β-d-arabinofuranosylcytosine in plasma to 1-β-d-arabinofuranosylcytosine 5′-triphosphate levels in leukemic cells during treatment with high-dose 1-β-d-arabinofuranosylcytosine

Cancer Res.

Metabolism of 1-β-d-arabinofuranosylcytosine in human leukemic cells

Cancer Res.

The relationship of ara-C metabolism in vitro to therapeutic response in acute myeloid leukaemia

Cancer Chemother. Pharmac.

In-vitro studies on phosphorylation and dephosphorylation of cytosine arabinoside in human leukemic cells

Leukemia Res.

Saturation of 1-β-d-arabinofuranosylcytosine 5′-triphosphate accumulation in leukemia cells during high-dose 1-β-d-arabinofuranosylcytosine therapy

Cancer Res.

Cited by (29)

Expression of high Km 5′-nucleotidase in leukemic blasts is an independent prognostic factor in adults with acute myeloid leukemia

Protection of leukemic cells by deoxycytidine: In vitro measures of protection against cytosine arabinoside

Nucleoside transport and its significance for anticancer drug resistance

Deoxycytidine in human plasma: Potential for protecting leukemic cells during chemotherapy

Effects of fludarabine and gemcitabine on human acute myeloid leukemia cell line HL 60: Direct comparison of cytotoxicity and cellular Ara-C uptake enhancement

Formation of cytosine arabinoside-5′-triphosphate in different cultured lymphoblastic leukaemic cells with reference to their drug sensitivity

ReviewSaturation of intracellular cytosine arabinoside triphosphate accumulation in human leukemic blast cells

Abstract

Blood

Blood

Blood

Analyt. Biochem.

High dose cytarabine: a review

Leukemia

Lethality of human myeloblasts correlates with the incorporation of arabinofuranosylcytosine into DNA

Relationship of 1-β-d-arabinofuranosylcytosine in plasma to 1-β-d-arabinofuranosylcytosine 5′-triphosphate levels in leukemic cells during treatment with high-dose 1-β-d-arabinofuranosylcytosine

Cancer Res.

Metabolism of 1-β-d-arabinofuranosylcytosine in human leukemic cells

Cancer Res.

The relationship of ara-C metabolism in vitro to therapeutic response in acute myeloid leukaemia

Cancer Chemother. Pharmac.

In-vitro studies on phosphorylation and dephosphorylation of cytosine arabinoside in human leukemic cells

Leukemia Res.

Saturation of 1-β-d-arabinofuranosylcytosine 5′-triphosphate accumulation in leukemia cells during high-dose 1-β-d-arabinofuranosylcytosine therapy

Cancer Res.

Review
Saturation of intracellular cytosine arabinoside triphosphate accumulation in human leukemic blast cells