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We have sought to clarify the potential activity of the S-phase-specific antileukemic agent 1-beta-D-arabinofuranosylcytosine (ara-C), an inhibitor of DNA synthesis, in quiescent cells that are substantially non-sensitive to nucleoside analogues. It was hypothesized that the combination of ara-C with DNA damaging agents that initiate DNA repair will expand ara-C cytotoxicity to non-cycling cells. The repair kinetics, which included incision of damaged DNA, gap-filling by DNA synthesis and rejoining by ligation, were evaluated using the single cell gel electrophoresis (Comet) assay and the thymidine incorporation assay. When normal lymphocytes were treated with ultraviolet C or with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), the processes of DNA excision repair were promptly initiated and rapidly completed. When the cells were incubated with ara-C prior to irradiation or BCNU treatment, the steps of DNA synthesis and rejoining in the repair processes were both inhibited. The ara-C-mediated inhibition of the repair processes was concentration-dependent, with the effect peaking at 10 microM. The combination of ara-C with these DNA repair initiators exerted subsequent cytotoxicity, which was proportional to the extent of the repair inhibition in the presence of ara-C. In conclusion, ara-C was cytotoxic in quiescent cells undergoing DNA repair. This might be attributed to unrepaired DNA damage that remained in the cells, thereby inducing lethal cytotoxicity. Alternatively, ara-C might exert its own cytotoxicity by inhibiting DNA synthesis in the repair processes. Such a strategy may be effective against a dormant subpopulation in acute leukemia that survives chemotherapy.

Alkylating agents or platinum analogues initiate several excision repair mechanisms, which involve incision of the DNA strand, excision of the damaged nucleotide, gap filling by DNA resynthesis, and rejoining by ligation. The previous study described that nucleotide excision repair permitted incorporation of fludarabine nucleoside (F‐ara‐A) into the repair patch, thereby inhibiting the DNA resynthesis. In the present study, to clarify the repair kinetics in view of the inhibition by F‐ara‐A, normal lymphocytes were stimulated to undergo nucleotide excision repair by ultraviolet C (UV) irradiation in the presence or absence of F‐ara‐A. The repair kinetics were determined as DNA single strand breaks resulting from the incision and the rejoining using the alkaline single cell gel electrophoresis (comet) assay. DNA resynthesis was evaluated in terms of the uptake of tritiated thymidine into DNA. The lymphocytes initiated the incision step maximally at 1 h, and completed the rejoining process within 4 h after UV exposure. UV also initiated thymidine uptake, which increased time‐dependently and reached a plateau at 4 h. A 2–h pre‐incubation with F‐ara‐A inhibited the repair in a concentration‐dependent manner, with the maximal inhibition by 5 μM. This inhibitory effect was demonstrated by the reduction of the thymidine uptake and by the inhibition of the rejoining. A DNA polymerase inhibitor, aphidicolin, and a ribonucleotide reductase inhibitor, hydroxyurea, were not so inhibitory to the repair process as F‐ara‐A at equimolar concentrations. The present findings suggest that inhibition of nucleotide excision repair may represent a novel therapeutic strategy against cancer, especially in the context of resistant cells with an increased repair capacity.

Glucocorticoids remain among the most important drugs in the treatment of acute lymphoblastic leukemia (ALL). Although the mechanisms of glucocorticoid resistance have been studied in some T‐cell leukemic cell lines, less work has been done with B‐cell lines. We established a dexamethasone (DEX)‐resistant human pre‐B lineage leukemia cell line (697/DEX) and investigated the mechanism of resistance. 697/DEX was over 430–fold more resistant to DEX compared with the parental cells (697/Neo). Overexpression of Bcl–2 protein was not observed in 697/DEX, different from the mechanism of resistance in Bcl–2–virus‐infected cells (697/Bcl–2). Although the expression of p‐glycoprotein (Pgp) in 697/DEX was positive, its functional activity was not detected. The numbers of glucocorticoid receptors (GR) in 697/DEX and 697/Bcl–2 were significantly lower than those in 697/Neo. In addition, 697/DEX and 697/Bcl–2 had higher levels of glutathione (GSH) than 697/Neo. In the presence of l‐buthionine‐(S, R)‐sulfoximine (BSO), an inhibitor of GSH synthesis, both 697/DEX and 697/Bcl–2 recovered their sensitivity to DEX. Interestingly, cell death by the depletion of GSH did not involve caspase–3/7 activation in 697/Bcl–2 and 697/DEX, different from 697/Neo, suggesting a death mechanism through caspase‐independent programmed cell death or necrosis. In conclusion, DEX‐resistance in 697/DEX was related not only to a GR decrease, but also to an increase in intracellular GSH level in the DEX‐resistant B‐cell leukemia cell line. Circumvention of DEX‐resistance with BSO may offer an approach to overcoming resistance to chemotherapy in B‐cell lineage ALL.

1-beta-D-Arabinofuranosylcytosine (ara-C) is used empirically at a low, conventional, or high dose. Ara-C therapy may be optimal if it is directed by the clinical pharmacokinetics of the intracellular active metabolite of ara-C, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP). However, ara-CTP has seldom been monitored during low- and conventional-dose ara-C therapies because detection methods were insufficiently sensitive. Here, with the use of our newly established method (Cancer Res., 56, 1800 -- 1804 (1996)), ara-CTP was monitored in leukemic cells from acute myelogenous leukemia patients receiving low- or conventional-dose ara-C [subcutaneous ara-C administration (10 mg / m(2) ) (3 patients), continuous ara-C infusion (20 or 70 mg / m(2) / 24 h) (7 patients), 2-h ara-C infusion (70 mg / m(2) ) (4 patients), and 2-h infusion of N(4)-behenoyl-1-beta-D-arabinofuranosylcytosine, a deaminase-resistant ara-C derivative (70 mg / m(2) ) (6 patients)]. Ara-CTP could be determined at levels under 1 microM. There was a close correlation between the elimination half-life values of the plasma ara-C and the intracellular ara-CTP. The presence of ara-C in the plasma was important to maintain ara-CTP. The continuous ara-C and the 2-h N(4)-behenoyl-1-beta-D-arabinofuranosylcytosine infusions maintained ara-CTP and the plasma ara-C longer than the subcutaneous ara-C or the 2-h ara-C infusion. They also afforded relatively higher ara-CTP concentrations, and consequently produced ara-CTP more efficiently than the 2-h ara-C infusion. Different administration methods produced different quantities of ara-CTP even at the same dose.

Complete elimination of the plasma cell dyscrasia is a rational therapeutic goal, as intercepting supply of precursor protein is a necessary condition for a major regression of amyloid deposits. High-dose melphalan with autologous stem cell transplantation has shown the ability to induce complete hematological response (HR) along with recovery of organ dysfunction. However, the rate of HR with this treatment rarely exceeds 40%. We describe here the first known case of successful reduced intensity allogeneic stem cell transplantation (RIST) for a patient with primary amyloidosis complicated with nephrotic syndrome but without cardiac disease, who had obtained only partial HR by high-dose melphalan with autologous stem cell transplantation. RIST may be feasible and be capable of achieving complete HR along with recovery from nephrotic syndrome with acceptable toxicity.

In L1210 cells incubated with 1-beta-D-arabinofuranosylcytosine (ara-C), 6-mercaptopurine (6-MP) significantly potentiated 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) accumulation and ara-C incorporation into DNA (ara-C/DNA). The cytotoxicity of these two drugs was assessed to be at least additive by clonogenic assay. 1-beta-D-Arabinofuranosyluracil (ara-U) level in a cell suspension was suppressed by 6-MP in a concentration-dependent fashion, though intracellular cytidine deaminase (CDD) activity was not affected by 6-MP. In addition, extracted CDD activity was not directly inhibited by 6-MP or by its intracellular metabolites in vitro. After preincubation in the presence or absence of 6-MP, the cell suspension was fractionated to obtain the spent medium and cell pellet. Then, each fraction was incubated with ara-C. Ara-U formation in the spent medium was found to increase conspicuously in relation to the time of preincubation in the control and it was suppressed by 6-MP pretreatment. Ara-U formation in the cell compartment increased slightly in relation to the time of preincubation in the control and substantially no suppression of ara-U formation was observed in spite of 6-MP pretreatment. In conclusion, intracellularly synthesized CDD was thought to be rapidly shed into the medium and the released CDD could play an important role in ara-C inactivation. 6-MP interrupted some step between synthesis and shedding of CDD, resulting in a decrease of the ara-C deamination in the medium and enhancement of its antileukemic effect.