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 IC 50 of 2–30 nM. Panobinostat also similarly inhibited the growth of all of the cell lines with an IC 50 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.

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 µ µ µ µL) were eluted isocratically using 0.06 M Na 2 HPO 4 (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 µ µ µ 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) G emcitabine (2′,2-difluorodeoxycytidine, dFdC) is the most important deoxycytidine analog to be developed since cytarabine: it has geminal fluorine atoms inserted at the 2′-carbon of the deoxyribofuranosyl ring.(1,2) The drug is active against not only leukemic cells in vitro but also several experimental solid tumors such as non-small-cell lung cancer, small-cell lung cancer and pancreatic cancer.(3-6) Clinically, dFdC has become the standard first-line therapy for patients with advanced pancreatic cancer, and it is rapidly becoming incorporated into first-line regimens in non-small-cell lung cancer and transitional-cell carcinoma of the bladder.(7-9) The efficacy of dFdC has also been evaluated for leukemic patients. (10,11) Thus, dFdC is now used widely for treating both solid tumors and hematological malignancies. (7)(8)(9)(10)(11) 2′,2-Difluorodeoxycytidine is a prodrug that requires intracellular activation.(1,2) After being transported into cancer cells, it is phosphorylated via dFdC monophosphate and diphosphate to dFdC triphosphate (dFdCTP), the process of which is catalyzed by several kinases including the rate-limiting enzyme deoxycytidine kinase. dFdCTP is incorporated into DNA at the penultimate position and, after the incorporation of one or more nucleotides, blocks further elon...

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.

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.

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 ...

Background Cytarabine (ara-C) is the major drug for the treatment of acute myeloid leukemia (AML), but cellular resistance to ara-C is a major obstacle to therapeutic success. The present study examined enhanced anti-apoptosis identified in 3 newly established nucleoside analogue-resistant leukemic cell line variants and approaches to overcoming this resistance. Methods HL-60 human AML cells were used to develop the ara-C– or clofarabine (CAFdA)-resistant variants. The Bcl-2 inhibitor venetoclax and the Mcl-1 inhibitor alvocidib were tested to determine whether they could reverse these cells’ resistance. Results A 10-fold ara-C-resistant HL-60 variant, a 4-fold CAFdA-resistant HL-60 variant, and a 30-fold CAFdA-resistant HL-60 variant were newly established. The variants demonstrated reduced deoxycytidine kinase and deoxyguanosine kinase expression, but intact expression of surface transporters (hENT1, hENT2, hCNT3). The variants exhibited lower expression of intracellular nucleoside analogue triphosphates compared with non-variant HL-60 cells. The variants also overexpressed Bcl-2 and Mcl-1. Venetoclax as a single agent was not cytotoxic to the resistant variants. Nevertheless, venetoclax with nucleoside analogs demonstrated synergistic cytotoxicity against the variants. Alvocidib as a single agent was cytotoxic to the cells. However, alvocidib induced G1 arrest and suppressed the cytotoxicity of the co-administered nucleoside analogs. Conclusions Three new nucleoside analogue-resistant HL-60 cell variants exhibited reduced production of intracellular analogue triphosphates and enhanced Bcl-2 and Mcl-1 expressions. Venetoclax combined with nucleoside analogs showed synergistic anti-leukemic effects and overcame the drug resistance.

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Background Pralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and explored potential therapeutic strategies to overcome PDX resistance. Methods To investigate PDX resistance, we established two PDX-resistant T-lymphoblastic leukemia cell lines (CEM and MOLT4) through continuous exposure to increasing doses of PDX. The resistance mechanisms were evaluated by measuring PDX uptake, apoptosis induction and folate metabolism-related protein expression. We also applied gene expression analysis and methylation profiling to identify the mechanisms of resistance. We then explored rational drug combinations using a spheroid (3D)-culture assay. Results Compared with their parental cells, PDX-resistant cells exhibited a 30-fold increase in half-maximal inhibitory concentration values. Induction of apoptosis by PDX was significantly decreased in both PDX-resistant cell lines. Intracellular uptake of [14C]-PDX decreased in PDX-resistant CEM cells but not in PDX-resistant MOLT4 cells. There was no significant change in expression of dihydrofolate reductase (DHFR) or folylpolyglutamate synthetase (FPGS). Gene expression array analysis revealed that DNA-methyltransferase 3β (DNMT3B) expression was significantly elevated in both cell lines. Gene set enrichment analysis revealed that adipogenesis and mTORC1 signaling pathways were commonly upregulated in both resistant cell lines. Moreover, CpG island hypermethylation was observed in both PDX resistant cells lines. In the 3D-culture assay, decitabine (DAC) plus PDX showed synergistic effects in PDX-resistant cell lines compared with parental lines. Conclusions The resistance mechanisms of PDX were associated with reduced cellular uptake of PDX and/or overexpression of DNMT3B. Epigenetic alterations were also considered to play a role in the resistance mechanism. The combination of DAC and PDX exhibited synergistic activity, and thus, this approach might improve the clinical efficacy of PDX.