Summary. Factors that reduce the intracellular concentration of triphosphorylated cytarabine (ara-CTP), the active form of cytarabine (ara-C), may induce chemoresistance in acute myeloid leukaemia (AML) patients. These factors include reduced influx of ara-C by the hENT1 transporter, reduced phosphorylation by deoxycytidine kinase (dCK), and increased degradation by high Km cytoplasmic 5¢-nucleotidase (5NT) and/or cytidine deaminase (CDD). Increased levels of DNA polymerase a (DNA POL) and reduced levels of topoisomerase I (TOPO I) and topoisomerase II (TOPO II) have also been detected in ara-C-resistant cell lines. To determine whether these factors are implicated in clinical ara-C resistance, we analysed the expression of these parameters at diagnosis, using reverse transcription polymerase chain reaction, in the blast cells of 123 AML patients treated with ara-C. At diagnosis, hENT1, dCK, CDD, 5NT, TOPO I, TOPO II, DNA POL and MDR1 were expressed in 83%, 22%, 7%, 37%, 59%, 37%, 39% and 16% of patients respectively. In univariate analysis, patients with expression of 5NT or DNA POL at diagnosis had significantly shorter disease-free survival (DFS). In multivariate analysis, DNA POL positivity and hENT1 deficiency were related to a shorter DFS. In univariate analysis, patients with 5NT-positive blasts had significantly shorter overall survival (OS). In multivariate analysis, shorter OS was related to DNA POL positivity. These results suggest that expression of DNA POL, 5NT and hENT1 at diagnosis may be resistance mechanisms to ara-C in AML patients.

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Summary. The cytotoxic activity of cytarabine (ara-C) in leukaemic blasts depends on activating enzymes such as deoxycytidine kinase (dCK) and inactivating enzymes such as the 5¢-nucleotidases. We have analysed dCK and 'highKm' 5¢-nucleotidase (cN-II) mRNA expression by the quantitative real-time polymerase chain reaction at diagnosis in leukaemic blasts from 115 acute myeloid leukaemia (AML) patients treated with ara-C. The prognostic value of these parameters as well as that of the cN-II/dCK ratio was determined. In univariate analyses: (1) low levels of dCK, high levels of cN-II and a high cN-II/dCK ratio predicted shorter disease-free survival (DFS); (2) low levels of dCK and cN-II/dCK ratio also predicted shorter overall survival (OS). In a multivariate analysis taking into account other clinical and laboratory variables: (1) high cN-II expression, a high cN-II/dCK ratio, age ‡ 60 years and an unfavourable karyotype were independent prognostic factors for DFS; and (2) a high cN-II/dCK ratio, age ‡ 60 years and an unfavourable karyotype predicted shorter OS. Age, karyotype and cN-II/dCK ratio were used to define a prognostic score that permitted the identification of high-and low-risk groups. Our results suggest that dCK and cN-II mRNA expression in leukaemic blasts at diagnosis is correlated with clinical outcome and may play a functional role in the resistance to ara-C in patients with AML.

B-cell chronic lymphocytic leukemia (B-CLL) is a malignant disease characterized by an accumulation of monoclonal CD5 þ mature B cells, with a high percentage of cells arrested in the G0/G1 phase of the cell cycle, and a particular resistance toward apoptosis-inducing agents. Dok1 (downstream of tyrosine kinases) is an abundant Ras-GTPase-activating protein (Ras-GAP)-associated tyrosine kinase substrate, which negatively regulates cell proliferation, downregulates MAP kinase activation and promotes cell migration. The gene encoding Dok1 maps to human chromosome 2p13, a region previously found to be rearranged in B-CLL. We have screened the Dok1 gene for mutations from 46 individuals with B-CLL using heteroduplex analysis. A four-nucleotide GGCC deletion in the coding region was found in the leukemia cells from one patient. This mutation causes a frameshift leading to protein truncation at the carboxyl-terminus, with the acquisition of a novel amino-acid sequence. In contrast to the wild-type Dok1 protein, which has cytoplasmic/ membrane localization, the mutant Dok1 is a nuclear protein containing a functional bipartite nuclear localization signal. Whereas overexpression of wild-type Dok1 inhibited PDGF-induced MAP kinase activation, this inhibition was not observed with the mutant Dok1. Furthermore the mutant Dok1 forms heterodimers with Dok1 wild type and the association can be enhanced by Lck-mediated tyrosine-phosphorylation. This is the first example of a Dok1 mutation in B-CLL and the data suggest that Dok1 might play a role in leukemogenesis.