Rie Nishi

Characterization of cytarabine-resistant leukemic cell lines established from five different blood cell lineages using gene expression and proteomic analyses

Cytarabine (ara-C) is the key drug for treatment of acute myeloid leukemia. Since intracellular cytarabine triphosphate (ara-CTP) is an active metabolite of ara-C, factors that reduce the amount of ara-CTP are known to induce drug resistance. However, these factors do not fully explain the development of resistance to ara-C. The present study was conducted to search for new candidate ara-C resistance factors, including those that are unrelated to ara-CTP production. For this purpose, we newly established five ara-C-resistant leukemic clones from different blood cell lineage leukemic cell lines (HL-60, K562, CEM, THP1 and U937). The resistant subclones were 5-58-fold more ara-C-resistant than their parental counterparts. All of the ara-C-resistant subclones, except for ara-C-resistant CEM cells, displayed alteration of ara-CTP-related factors such as ara-C membrane transport capacity, deoxycytidine kinase activity or cytosolic nucleotidase II activity. To identify new candidate factors, we used two comprehensive approaches: DNA microarray and proteome analyses. The DNA microarray analysis revealed eight genes (C19orf2, HSPA8, LGALS1, POU4F3, PSAP, AKT1, MBC2 and CACNA2D3) that were altered in all five ara-C-resistant lines compared to parental cells. Both proteome and DNA microarray analyses further detected a reduced protein level of stathmin1 in the ara-C-resistant CEM subclone compared to its parental line. Thus, the present findings suggested the involvement of novel multiple mechanisms in mediating the ara-C resistance of leukemic cells. The role of some of these molecules in resistance is still unclear.

A new, simple method for quantifying gemcitabine triphosphate in cancer cells using isocratic high‐performance liquid chromatography

A deoxycytidine analog, gemcitabine (dFdC), is effective for treating solid tumors and hematological malignancies. After being transported into cancer cells, dFdC is phosphorylated to dFdC triphosphate (dFdCTP), which is subsequently incorporated into the DNA strand, thereby inhibiting DNA synthesis. Intracellular dFdCTP is the critical determinant for dFdC cytotoxicity, so therapeutic drug monitoring or in vitro testing of the capability of cancer cells to accumulate dFdCTP may be informative for optimizing dFdC administration. We have developed a new isocratic‐elution high‐performance liquid chromatography method for quantifying dFdCTP in cancer cells. Samples (500 µL) were eluted isocratically using 0.06 M Na2HPO4 (pH 6.9) containing 20% acetonitrile, at a constant flow rate of 0.7 mL/min and at ambient temperature. Separation was carried out using an anion‐exchange column (TSK gel DEAE‐2SW; 250 mm × 4.6 mm inside diameter, particle size 5 µL) and monitored at 254 nm. The standard curve was linear with low within‐day and interday variability. The lower detection limit (20 pmol) was as sensitive as that of the previous gradient‐elution method. dFdCTP was well separated from other nucleoside triphosphates. The method could measure dFdCTP in cultured or primary leukemic cells treated in vitro with dFdC. The method was also applicable to simultaneous determination of dFdCTP and cytarabine triphosphate, the results of which demonstrated ara‐CTP production augmented by dFdC pretreatment. Thus, our isocratic high‐performance liquid chromatography assay method will be of great use because of its sensitivity and simplicity as well as its applicability to biological materials. (Cancer Sci 2006; 97: 1274–1278)

Reduced drug incorporation into DNA and antiapoptosis as the crucial mechanisms of resistance in a novel nelarabine-resistant cell line.

BackgroundNine-beta-D-arabinofuranosylguanine (ara-G), an active metabolite of nelarabine, enters leukemic cells through human Equilibrative Nucleoside Transporter 1, and is then phosphorylated to an intracellular active metabolite ara-G triphosphate (ara-GTP) by both cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. Ara-GTP is subsequently incorporated into DNA, thereby inhibiting DNA synthesis.MethodsIn the present study, we developed a novel ara-G-resistant variant (CEM/ara-G) of human T-lymphoblastic leukemia cell line CCRF-CEM, and elucidated its mechanism of ara-G resistance. The cytotoxicity was measured by using the growth inhibition assay and the induction of apoptosis. Intracellular triphosphate concentrations were quantitated by using HPLC. DNA synthesis was evaluated by the incorporation of tritiated thymidine into DNA. Protein expression levels were determined by using Western blotting.ResultsCEM/ara-G cells were 70-fold more ara-G-resistant than were CEM cells. CEM/ara-G cells were also refractory to ara-G-mediated apoptosis. The transcript level of human Equilibrative Nucleoside Transporter 1 was lowered, and the protein levels of deoxycytidine kinase and deoxyguanosine kinase were decreased in CEM/ara-G cells. The subsequent production of intracellular ara-GTP (21.3 pmol/107 cells) was one-fourth that of CEM cells (83.9 pmol/107 cells) after incubation for 6 h with 10 μM ara-G. Upon ara-G treatment, ara-G incorporation into nuclear and mitochondrial DNA resulted in the inhibition of DNA synthesis of both fractions in CEM cells. However, DNA synthesis was not inhibited in CEM/ara-G cells due to reduced ara-G incorporation into DNA. Mitochondrial DNA-depleted CEM cells, which were generated by treating CEM cells with ethidium bromide, were as sensitive to ara-G as CEM cells. Anti-apoptotic Bcl-xL was increased and pro-apoptotic Bax and Bad were reduced in CEM/ara-G cells.ConclusionsAn ara-G-resistant CEM variant was successfully established. The mechanisms of resistance included reduced drug incorporation into nuclear DNA and antiapoptosis.

Combination of panobinostat with ponatinib synergistically overcomes imatinib‐resistant CML cells

The major mechanism of imatinib (IM) resistance of CML is the reactivation of ABL kinase either through BCR‐ABL gene amplification or mutation. We investigated the cytotoxicity of a pan‐ABL tyrosine kinase inhibitor, ponatinib, and a pan‐histone deacetylase inhibitor, panobinostat, against IM‐resistant CML cells in vitro. Two different IM‐resistant cell lines, K562/IM‐R1 and Ba/F3/T315I were evaluated in comparison with their respective, parental cell lines, K562 and Ba/F3. K562/IM‐R1 overexpressed BCR‐ABL due to gene amplification. Ba/F3/T315I was transfected with a BCR‐ABL gene encoding T315I‐mutated BCR‐ABL. Ponatinib inhibited the growth of both K562/IM‐R1 and Ba/F3/T315I as potently as it inhibited their parental cells with an IC50 of 2–30 nM. Panobinostat also similarly inhibited the growth of all of the cell lines with an IC50 of 40–51 nM. This was accompanied by reduced histone deacetylase activity, induced histone H3 acetylation, and an increased protein level of heat shock protein 70, which suggested disruption of heat shock protein 90 chaperone function for BCR‐ABL and its degradation. Importantly, the combination of ponatinib with panobinostat showed synergistic growth inhibition and induced a higher level of apoptosis than the sum of the apoptosis induced by each agent alone in all of the cell lines. Ponatinib inhibited phosphorylation not only of BCR‐ABL but also of downstream signal transducer and activator of transcription 5, protein kinase B, and ERK1/2 in both K562/IM‐R1 and Ba/F3/T315I, and the addition of panobinostat to ponatinib further inhibited these phosphorylations. In conclusion, panobinostat enhanced the cytotoxicity of ponatinib towards IM‐resistant CML cells including those with T315I‐mutated BCR‐ABL.

Combination of guanine arabinoside and Bcl‐2 inhibitor YC137 overcomes the cytarabine resistance in HL‐60 leukemia cell line

Cytarabine (ara‐C) is the key agent for treating acute myeloid leukemia. After being transported into leukemic cells, ara‐C is phosphorylated, by several enzymes including deoxycytidine kinase (dCK), to ara‐C triphosphate (ara‐CTP), an active metabolite, and then incorporated into DNA, thereby inhibiting DNA synthesis. Therefore, the cytotoxicity of ara‐C depends on the production of ara‐CTP and the induction of apoptosis. Here, we established a new ara‐C‐resistant acute myeloid leukemia cell line (HL‐60/ara‐C60) with dual resistance characteristics of the anti‐antimetabolic character of decreased ara‐CTP production and an increase in the antiapoptotic factors Bcl‐2 and Bcl‐XL. We further attempted to overcome resistance by augmenting ara‐CTP production and stimulating apoptosis. A relatively new nucleoside analog, 9‐β‐d‐arabinofuranosylguanine (ara‐G), and the small molecule Bcl‐2 antagonist YC137 were used for this purpose. HL‐60/ara‐C60 was 60‐fold more ara‐C‐resistant than the parental HL‐60 cells. HL‐60/ara‐C60 cells exhibited low dCK protein expression, which resulted in decreased ara‐CTP production. HL‐60/ara‐C60 cells were also refractory to ara‐C‐induced apoptosis due to overexpression of Bcl‐2 and Bcl‐XL. Combination treatment of ara‐C with ara‐G augmented the dCK protein level, thereby increasing ara‐CTP production and subsequent cytotoxicity. Moreover, the combination of ara‐C with YC137 produced a greater amount of apoptosis than ara‐C alone. Importantly, the three‐drug combination of ara‐C, ara‐G and YC137 provided greater cytotoxicity than ara‐C+ara‐G or ara‐C+YC137. These findings suggest possible combination strategies for overcoming ara‐C resistance by augmenting ara‐CTP production and reversing refractoriness against the induction of apoptosis in ara‐C resistant leukemic cells.

Quantitative Estimation of Urate Transport in Nephrons in Relation to Urinary Excretion Employing Benzbromarone-Loading Urate Clearance Tests in Cases of Hyperuricemia

Background: A four-component system for urate transport in nephrons has been proposed and widely investigated by various investigators studying the mechanisms underlying urinary urate excretion. However, quantitative determinations of urate transport have not been clearly elucidated yet. Methods: The equation C_ua = {C_cr(1 – R₁) + TSR}(1 – R₂) was designed to approximate mathematically urate transport in nephrons, where R₁ = urate reabsorption ratio; R₂ = urate postsecretory reabsorption ratio; TSR = tubular secretion rate; C_ua = urate clearance, and C_cr = creatinine clearance . To investigate relationships between the three unknown variables (R₁, R₂, and TSR), this equation was expressed as contour lines of one unknown on a graph of the other two unknowns. Points at regular intervals on each contour line for the equation were projected onto a coordinate axis and the high-density regions corresponding to high-density intervals of a coordinate were investigated for three graph types. For benzbromarone (BBR)-loading C_ua tests, C_ua was determined before and after oral administration of 100 mg of BBR and C_uaBBR(∞) was calculated from the ratio of C_uaBBR(100)/C_ua. Results: Before BBR administration, points satisfying the equation on the contour line for R₁ = 0.99 were highly dense in the region R₂ = 0.87–0.92 on all three graphs, corresponding to a TSR of 40–60 ml/min in hyperuricemia cases (HU). After BBR administration, the dense region was shifted in the direction of reductions in both R₁ and R₂, but TSR was unchanged. Under the condition that R₁ = 1 and R₂ = 0, urate tubular secretion (UTS) was considered equivalent to calculated urinary urate excretion (U_ex) in a model of intratubular urate flow with excess BBR; C_uaBBR(∞) = TSR was deduced from the equation at R₁ = 1 and R₂ = 0. In addition, TSR of the point under the condition that R₁ = 1 and R₂ = 0 on the graph agreed with TSR for the dense region at excess BBR. TSR was thus considered approximately equivalent to C_uaBBR(∞), which could be determined from a BBR-loading C_ua test. Approximate values for urate glomerular filtration, urate reabsorption, UTS, urate postsecretory reabsorption (UR₂), and U_ex were calculated as 9,610; 9,510; 4,490; 4,150, and 440 µg/min for HU and 6,890; 6,820; 4,060; 3,610, and 520 µg/min for normal controls (NC), respectively. The most marked change in HU was the decrease in TSR (32.0%) compared to that in NC, but UTS did not decrease. Calculated intratubular urate contents were reduced more by higher UR₂ in HU than in NC. This enhanced difference resulted in a 15.4% decrease in U_ex for HU. Conclusion: Increased UR₂ may represent the main cause of urate underexcretion in HU.