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The human equilibrative nucleoside transporter 1 mediates in vitro cytarabine sensitivity in childhood acute myeloid leukaemia.

Hubeek I, Stam RW, Peters GJ, Broekhuizen R, Meijerink JP, van Wering ER, Gibson BE, Creutzig U, Zwaan CM, Cloos J, Kuik DJ, Pieters R, Kaspers GJ - Br. J. Cancer (2005)

Bottom Line: Expression of the inactivating enzyme pyrimidine nucleotidase-I (PN-I) was 1.8-fold lower in FAB-M5 as compared to FAB-M1/2 (P=0.007).Human equilibrative nucleoside transporter-1 (hENT1) mRNA expression and ara-C sensitivity were significantly correlated (rp=-0.46; P=0.001), with three-fold lower hENT1 mRNA levels in resistant patients (P=0.003). hENT1 mRNA expression also seemed to correlate inversely with the LC50 values of cladribine (rp=-0.30; P=0.04), decitabine (rp=-0.29; P=0.04) and gemcitabine (rp=-0.33; P=0.02).In conclusion, decreased expression of hENT1, which transports ara-C across the cell membrane, appears to be a major factor in ara-C resistance in childhood AML.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Hematology/Oncology, VU University Medical Center, De Boelelaan 1117, Postbus 7057, 1007 MB, Amsterdam, The Netherlands. isabelle.hubeek@vumc.nl

ABSTRACT
Cytarabine (ara-C) is the most effective agent for the treatment of acute myeloid leukaemia (AML). Aberrant expression of enzymes involved in the transport/metabolism of ara-C could explain drug resistance. We determined mRNA expression of these factors using quantitative-real-time-PCR in leukemic blasts from children diagnosed with de novo AML. Expression of the inactivating enzyme pyrimidine nucleotidase-I (PN-I) was 1.8-fold lower in FAB-M5 as compared to FAB-M1/2 (P=0.007). In vitro sensitivity to deoxynucleoside analogues was determined using the MTT-assay. Human equilibrative nucleoside transporter-1 (hENT1) mRNA expression and ara-C sensitivity were significantly correlated (rp=-0.46; P=0.001), with three-fold lower hENT1 mRNA levels in resistant patients (P=0.003). hENT1 mRNA expression also seemed to correlate inversely with the LC50 values of cladribine (rp=-0.30; P=0.04), decitabine (rp=-0.29; P=0.04) and gemcitabine (rp=-0.33; P=0.02). Deoxycytidine kinase (dCK) and cytidine deaminase (CDA) mRNA expression seemed to correlate with in vitro sensitivity to gemcitabine (rp=-0.31; P=0.03) and decitabine (rp=0.33; P=0.03), respectively. The dCK/PN-I ratio correlated inversely with LC50 values for gemcitabine (rp=-0.45, P=0.001) and the dCK/CDA ratio seemed to correlate with LC50 values for decitabine (rp=-0.29; 0.04). In conclusion, decreased expression of hENT1, which transports ara-C across the cell membrane, appears to be a major factor in ara-C resistance in childhood AML.

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Metabolism of ara-C. Ara-C enters the cell via the equilibrative nucleoside transporter 1 (hENT1; 1). Inside the cell, ara-C is phosphorylated to ara-CMP by deoxycytidine kinase (dCK; 2). Ara-CMP is subsequently phosphorylated to ara-CTP, the active metabolite. Incorporation of ara-CTP into the DNA blocks DNA synthesis and leads to cell death. Ara-CTP formation can be obstructed. Pyrimidine nucleotidase I (PN-I; 3) opposes the action of dCK. Cytidine deaminase (CDA; 4) and deoxycytidylate deaminase (dCMPD; 5) convert ara-C to ara-U, and ara-CMP to ara-UMP, respectively. Increased intracellular dCTP pools antagonise the formation of ara-CTP. dCTP can be synthesised directly via the de novo pathway by ribonucleotide reductase (6). CTP synthetase (CTPs; 7) converts uridine triphosphate to CTP. Because aberrant expression of these enzymes may be related to in vitro sensitivity to ara-C, and other deoxynucleoside analogues, we determined the mRNA expression of the target genes in AML.
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fig1: Metabolism of ara-C. Ara-C enters the cell via the equilibrative nucleoside transporter 1 (hENT1; 1). Inside the cell, ara-C is phosphorylated to ara-CMP by deoxycytidine kinase (dCK; 2). Ara-CMP is subsequently phosphorylated to ara-CTP, the active metabolite. Incorporation of ara-CTP into the DNA blocks DNA synthesis and leads to cell death. Ara-CTP formation can be obstructed. Pyrimidine nucleotidase I (PN-I; 3) opposes the action of dCK. Cytidine deaminase (CDA; 4) and deoxycytidylate deaminase (dCMPD; 5) convert ara-C to ara-U, and ara-CMP to ara-UMP, respectively. Increased intracellular dCTP pools antagonise the formation of ara-CTP. dCTP can be synthesised directly via the de novo pathway by ribonucleotide reductase (6). CTP synthetase (CTPs; 7) converts uridine triphosphate to CTP. Because aberrant expression of these enzymes may be related to in vitro sensitivity to ara-C, and other deoxynucleoside analogues, we determined the mRNA expression of the target genes in AML.

Mentions: In combination with anthracyclines, 1-β-D-arabinofuranosylcytosine (cytosine arabinoside, cytarabine, ara-C) is the most effective agent for the treatment of AML. Ara-C is a deoxynucleoside analogue that has to be converted into its active triphosphate derivative (ara-CTP) to exert its cytotoxic effect (Grant, 1998). Ara-CTP is then incorporated into the DNA causing chain termination, resulting in a block in DNA synthesis and facilitating programmed cell death (Figure 1). Ara-C is a hydrophilic molecule and as such requires facilitated diffusion via nucleoside-specific membrane transport carriers to enter cells (Cass et al, 1998; Clarke et al, 2002). The human equilibrative nucleoside transporter (hENT1) is responsible for 80% of ara-C influx in human leukemic blast cells (Sundaram et al, 2001; Clarke et al, 2002). Inside the cell, conversion of ara-C into ara-CMP by deoxycytidine kinase (dCK) is believed to be the rate-limiting step in the metabolism of ara-C (Liliemark et al, 1985; Plunkett et al, 1987). Subsequently, ara-CMP is phosphorylated into ara-CDP by nucleoside monophosphate kinases, which in turn finally is phosphorylated into ara-CTP by diphosphate kinases (Hande and Chabner, 1978). Inactivation of ara-C results from deamination by cytidine deaminase (CDA) (Laliberte and Momparler, 1994). In addition, ara-CMP can be dephosphorylated by pyrimidine nucleotidase I (PN-I) (Amici et al, 1997) as well as deaminated by deoxycytidylate deaminase (dCMPD) (Mancini and Cheng, 1983). Inactivation by these enzymes decreases the amount of ara-CTP and thereby limits ara-C mediated cytotoxicity. Deoxycytidine triphosphate (dCTP) is thought to inhibit the phosphorylation of ara-C (by feedback inhibition of dCK) and the incorporation of ara-CTP into DNA (by competition for DNA polymerase). Increased intracellular dCTP pools therefore antagonise the formation of ara-CTP (Liliemark and Plunkett, 1986; White and Capizzi, 1991). dCTP can be synthezised directly via the de novo pathway by ribonucleotide reductase (RR) (Smith and Karp, 2003). Ribonucleotide reductase catalyses the conversion of ribonucleotides into deoxyribonucleotides (Smith and Karp, 2003). Mammalian RR is made up of two subunits (Reichard and Ehrenberg, 1983). The M1 subunit is the binding site for nucleotides and the second subunit, M2, is a metal binding site that requires both a non-haeme iron and a tyrosine-free radical for its activity (Smith and Karp, 2003). CTP synthetase (CTPs) is responsible for the conversion of uridine triphosphate (UTP) into CTP and has a high activity in several malignancies, including acute lymphoblastic leukaemia (Verschuur et al, 1998).


The human equilibrative nucleoside transporter 1 mediates in vitro cytarabine sensitivity in childhood acute myeloid leukaemia.

Hubeek I, Stam RW, Peters GJ, Broekhuizen R, Meijerink JP, van Wering ER, Gibson BE, Creutzig U, Zwaan CM, Cloos J, Kuik DJ, Pieters R, Kaspers GJ - Br. J. Cancer (2005)

Metabolism of ara-C. Ara-C enters the cell via the equilibrative nucleoside transporter 1 (hENT1; 1). Inside the cell, ara-C is phosphorylated to ara-CMP by deoxycytidine kinase (dCK; 2). Ara-CMP is subsequently phosphorylated to ara-CTP, the active metabolite. Incorporation of ara-CTP into the DNA blocks DNA synthesis and leads to cell death. Ara-CTP formation can be obstructed. Pyrimidine nucleotidase I (PN-I; 3) opposes the action of dCK. Cytidine deaminase (CDA; 4) and deoxycytidylate deaminase (dCMPD; 5) convert ara-C to ara-U, and ara-CMP to ara-UMP, respectively. Increased intracellular dCTP pools antagonise the formation of ara-CTP. dCTP can be synthesised directly via the de novo pathway by ribonucleotide reductase (6). CTP synthetase (CTPs; 7) converts uridine triphosphate to CTP. Because aberrant expression of these enzymes may be related to in vitro sensitivity to ara-C, and other deoxynucleoside analogues, we determined the mRNA expression of the target genes in AML.
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Related In: Results  -  Collection

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fig1: Metabolism of ara-C. Ara-C enters the cell via the equilibrative nucleoside transporter 1 (hENT1; 1). Inside the cell, ara-C is phosphorylated to ara-CMP by deoxycytidine kinase (dCK; 2). Ara-CMP is subsequently phosphorylated to ara-CTP, the active metabolite. Incorporation of ara-CTP into the DNA blocks DNA synthesis and leads to cell death. Ara-CTP formation can be obstructed. Pyrimidine nucleotidase I (PN-I; 3) opposes the action of dCK. Cytidine deaminase (CDA; 4) and deoxycytidylate deaminase (dCMPD; 5) convert ara-C to ara-U, and ara-CMP to ara-UMP, respectively. Increased intracellular dCTP pools antagonise the formation of ara-CTP. dCTP can be synthesised directly via the de novo pathway by ribonucleotide reductase (6). CTP synthetase (CTPs; 7) converts uridine triphosphate to CTP. Because aberrant expression of these enzymes may be related to in vitro sensitivity to ara-C, and other deoxynucleoside analogues, we determined the mRNA expression of the target genes in AML.
Mentions: In combination with anthracyclines, 1-β-D-arabinofuranosylcytosine (cytosine arabinoside, cytarabine, ara-C) is the most effective agent for the treatment of AML. Ara-C is a deoxynucleoside analogue that has to be converted into its active triphosphate derivative (ara-CTP) to exert its cytotoxic effect (Grant, 1998). Ara-CTP is then incorporated into the DNA causing chain termination, resulting in a block in DNA synthesis and facilitating programmed cell death (Figure 1). Ara-C is a hydrophilic molecule and as such requires facilitated diffusion via nucleoside-specific membrane transport carriers to enter cells (Cass et al, 1998; Clarke et al, 2002). The human equilibrative nucleoside transporter (hENT1) is responsible for 80% of ara-C influx in human leukemic blast cells (Sundaram et al, 2001; Clarke et al, 2002). Inside the cell, conversion of ara-C into ara-CMP by deoxycytidine kinase (dCK) is believed to be the rate-limiting step in the metabolism of ara-C (Liliemark et al, 1985; Plunkett et al, 1987). Subsequently, ara-CMP is phosphorylated into ara-CDP by nucleoside monophosphate kinases, which in turn finally is phosphorylated into ara-CTP by diphosphate kinases (Hande and Chabner, 1978). Inactivation of ara-C results from deamination by cytidine deaminase (CDA) (Laliberte and Momparler, 1994). In addition, ara-CMP can be dephosphorylated by pyrimidine nucleotidase I (PN-I) (Amici et al, 1997) as well as deaminated by deoxycytidylate deaminase (dCMPD) (Mancini and Cheng, 1983). Inactivation by these enzymes decreases the amount of ara-CTP and thereby limits ara-C mediated cytotoxicity. Deoxycytidine triphosphate (dCTP) is thought to inhibit the phosphorylation of ara-C (by feedback inhibition of dCK) and the incorporation of ara-CTP into DNA (by competition for DNA polymerase). Increased intracellular dCTP pools therefore antagonise the formation of ara-CTP (Liliemark and Plunkett, 1986; White and Capizzi, 1991). dCTP can be synthezised directly via the de novo pathway by ribonucleotide reductase (RR) (Smith and Karp, 2003). Ribonucleotide reductase catalyses the conversion of ribonucleotides into deoxyribonucleotides (Smith and Karp, 2003). Mammalian RR is made up of two subunits (Reichard and Ehrenberg, 1983). The M1 subunit is the binding site for nucleotides and the second subunit, M2, is a metal binding site that requires both a non-haeme iron and a tyrosine-free radical for its activity (Smith and Karp, 2003). CTP synthetase (CTPs) is responsible for the conversion of uridine triphosphate (UTP) into CTP and has a high activity in several malignancies, including acute lymphoblastic leukaemia (Verschuur et al, 1998).

Bottom Line: Expression of the inactivating enzyme pyrimidine nucleotidase-I (PN-I) was 1.8-fold lower in FAB-M5 as compared to FAB-M1/2 (P=0.007).Human equilibrative nucleoside transporter-1 (hENT1) mRNA expression and ara-C sensitivity were significantly correlated (rp=-0.46; P=0.001), with three-fold lower hENT1 mRNA levels in resistant patients (P=0.003). hENT1 mRNA expression also seemed to correlate inversely with the LC50 values of cladribine (rp=-0.30; P=0.04), decitabine (rp=-0.29; P=0.04) and gemcitabine (rp=-0.33; P=0.02).In conclusion, decreased expression of hENT1, which transports ara-C across the cell membrane, appears to be a major factor in ara-C resistance in childhood AML.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Hematology/Oncology, VU University Medical Center, De Boelelaan 1117, Postbus 7057, 1007 MB, Amsterdam, The Netherlands. isabelle.hubeek@vumc.nl

ABSTRACT
Cytarabine (ara-C) is the most effective agent for the treatment of acute myeloid leukaemia (AML). Aberrant expression of enzymes involved in the transport/metabolism of ara-C could explain drug resistance. We determined mRNA expression of these factors using quantitative-real-time-PCR in leukemic blasts from children diagnosed with de novo AML. Expression of the inactivating enzyme pyrimidine nucleotidase-I (PN-I) was 1.8-fold lower in FAB-M5 as compared to FAB-M1/2 (P=0.007). In vitro sensitivity to deoxynucleoside analogues was determined using the MTT-assay. Human equilibrative nucleoside transporter-1 (hENT1) mRNA expression and ara-C sensitivity were significantly correlated (rp=-0.46; P=0.001), with three-fold lower hENT1 mRNA levels in resistant patients (P=0.003). hENT1 mRNA expression also seemed to correlate inversely with the LC50 values of cladribine (rp=-0.30; P=0.04), decitabine (rp=-0.29; P=0.04) and gemcitabine (rp=-0.33; P=0.02). Deoxycytidine kinase (dCK) and cytidine deaminase (CDA) mRNA expression seemed to correlate with in vitro sensitivity to gemcitabine (rp=-0.31; P=0.03) and decitabine (rp=0.33; P=0.03), respectively. The dCK/PN-I ratio correlated inversely with LC50 values for gemcitabine (rp=-0.45, P=0.001) and the dCK/CDA ratio seemed to correlate with LC50 values for decitabine (rp=-0.29; 0.04). In conclusion, decreased expression of hENT1, which transports ara-C across the cell membrane, appears to be a major factor in ara-C resistance in childhood AML.

Show MeSH
Related in: MedlinePlus