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Background: The development of newer strategies to improve outcomes for older patients with secondary acute myeloid leukemia (s-AML) is a critical unmet need. Establishing baseline metrics for evaluating newer approaches is important., Methods: s-AML was defined as 1 or more of the following: a history of an antecedent hematologic disorder (AHD), a diagnosis of therapy-related acute myeloid leukemia (AML), and AML with karyotype abnormalities characteristic of myelodysplastic syndrome. Newly diagnosed s-AML patients aged 60 to 75 years were grouped into 5 treatment cohorts: 1) patients receiving high- or intermediate-dose cytarabine-based intensive chemotherapy (IC), 2) patients receiving a hypomethylating agent (HMA) or HMA combinations, 3) patients receiving low-dose cytarabine (LDAC) combinations, 4) patients receiving CPX-351, and 5) patients receiving investigational (INV) agents. Nine hundred thirty-one patients met the age and s-AML criteria., Results: Complete remission rates were statistically lower in the HMA group (36%) versus the IC (46%), CPX-351 (45%), and LDAC groups (43%). Patients receiving less intensive regimens (the HMA and LDAC groups combined) had superior overall survival (OS) in comparison with patients receiving IC-based regimens (median 6.9 vs 5.4 months; P = .048). Only 4.3% of the IC patients proceeded to transplantation, whereas 10.3% of the patients on lower intensity regimens did (P = .001). There was no difference in median survival between patients treated with CPX-351 and patients treated with conventional lower intensity approaches (P = .75). Age > 70 years, an adverse karyotype, and a prior AHD were associated with decreased OS in a multivariate analysis., Conclusions: Lower intensity approaches are associated with lower early mortality rates and improved OS in comparison with intensive regimens. OS is poor with currently available therapies with a median OS of 6 months (5.4-7.6 months across regimens). Unsatisfactory outcomes with other INV agents underscore the need for more effective therapies. Cancer 2017;123:3050-60. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Background: High-dose cytarabine (HiDAC) is safe and very effective in younger patients with acute myeloid leukemia (AML), but it generally is not well tolerated in the elderly., Methods: The authors explored the safety and tolerability of a modified HiDAC induction regimen consisting of 6 daily doses of cytarabine at 2 g/m(2) in combination with 3 daily doses of daunorubicin at 45 mg/m(2) in 59 consecutive patients aged >60 years who had de novo AML diagnosed between July 1996 and February 2005., Results: The median patient age was 68 years (range, 60-86 years). The regimen was well tolerated. Infections were common and occurred in 39% of patients, but cerebellar toxicities occurred in only 7% of patients and were reversible. The day-30 induction-related mortality rate was 10%. Overall, 69% of patients achieved complete remissions (CR), and 80% received up to 3 consolidations with HiDAC. The median follow-up for surviving patients was 53 months (range, 17-114 months). The median overall survival was 15.3 months (range, 1-114 months), and the relapse-free survival was 13.8 months (range, 1-113 months). Survival for patients who achieved CR was 27 months (range, 2-114 months)., Conclusions: The modified HiDAC regimen was well tolerated in patients aged >60 years with AML and was associated with low induction mortality and high rates of CR. Nevertheless, these high remissions still were associated with poor overall outcomes., (Copyright © 2011 American Cancer Society.)

Human granulocyte-macrophage colony-stimulating factor fused to truncated diphtheria toxin (DT388-GM-CSF) sensitized wild-type and Bcl2-overexpressing HL60 human leukemia cells to intoxication by Ara-C based on proliferation and clonogenic assays. The toxin/drug combination showed dramatic synergistic toxicity with combination indices of < 0.1. Synergy was not seen with two other protein synthesis inhibiting drugs--ricin and cycloheximide nor with GMCSF alone. No changes in Ara-C incorporation into cellular DNA or cell cycle occupancy were seen. As compared to exposure to DT388-GM-CSF or Ara-C alone, co-treatment produced significant increases in cytosolic accumulation of cytochrome c, a higher percentage of cells with loss of mitochondrial membrane potential and an increase in reactive oxygen species and morphologic changes of apoptosis, and a greater induction of poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor 45 (DFF45) cleavage activities of caspase 3. Co-treatment did not significantly alter Bcl2, Bcl-xL, Bax or Fas receptor (FasR), but modestly increased Fas ligand (FasL) protein. These finding suggest that co-treatment with DT388-GM-CSF may lead to a lowered apoptotic threshold and clonogenic survival of human AML blasts due to Ara-C. These observations also suggest that clinical trials of combination therapy may be warranted in patients with AML.

Human chronic myelogenous leukemia-blast crisis K562 cells have been demonstrated to be relatively resistant to antileukemic drug-induced apoptosis. This has been attributed to the activity of p210bcr-abl tyrosine kinase present in the K562 cells, which is known to suppress drug-induced apoptosis. Recently, K562 cells have been shown to express the antiapoptosis Bcl-xL but not Bcl-2 proteins. To investigate the contribution of Bcl-xL toward resistance to drug-induced apoptosis, we created K562/Bcl-xS and K562/neo cells by electroporating the expression plasmids pSFFVneo-Bcl-xS and pSFFVneo, containing the bcl-xS and neomycin resistance genes, respectively, into K562 cells. K562/Bcl-xS but not K562/neo cells expressed the bcl-xS mRNA and p19Bcl-xS protein. In contrast, both cell types expressed equivalent levels of Bcl-xL, Bax, Bcl-2, Myc, retinoblastoma, p21cbor-abl, and p145abl proteins. A significant increase in the hemoglobin levels was observed in the K562/Bcl-xS compared with the K562/neo cells (P < 0.05). In addition, K562/Bcl-xS cells were significantly more sensitive than K562/neo cells to undergoing erythroid differentiation induced by low-dose 1-beta-D-arabinofuranosylcytosine (ara-C) and hexamethyl bisacetamide (P < 0.05), but not by all-trans-retinoic acid. Low-dose ara-C- or hexamethyl bisacetamide-induced differentiation was not associated with apoptosis of K562/Bcl-xS or K562/neo cells. Low-dose ara-C-induced erythroid differentiation was accompanied by conversion of the retinoblastoma protein to predominantly its underphosphorylated isoform as well as by down-regulation of Myc levels in K562/Bcl-xS and K562/neo cells. Importantly, exposure to high-dose ara-C (HIDAC; 100 microM ara-C for 4 h) caused internucleosomal DNA fragmentation and the morphological features of apoptosis in K562/Bcl-xS cells. These effects were modestly enhanced by cotreatment with HIDAC plus herbimycin A. In contrast, K562/neo cells were completely resistant to HIDAC- and herbimycin A-induced apoptosis. These results indicate that the expression of Bcl-xS induces erythroid differentiation and partially sensitizes chronic myelogenous leukemia-blast crisis-derived K562 cells to ara-C-induced differentiation and apoptosis.

We examined the effects of high intracellular levels of Bcl-2 on the metabolism and DNA incorporation of high-dose Ara-C (HIDAC) as well as on Ara-C-induced DNA strand breaks and apoptosis of human AML HL-60 cells. HL-60/Bcl-2 and HL-60/neo cells were created by retrovirally transfecting the human AML HL-60 cells with the pZip-bcl-2 and pZip-neo plasmids, respectively. As compared to HL-60/neo, HL-60/Bcl-2 cells contained significantly higher (approximately 10-fold) p26Bcl-2, but equivalent levels of Bax and undetectable levels of Bcl-xL. HIDAC (10 or 100 microM for 4 h) produced the kilobase size and internucleosomal DNA fragmentation associated with apoptosis in HL-60/neo but not in HL-60/Bcl-2 cells. Significantly greater loss of survival (by MTT assay) and flowcytometric and morphologically recognizable apoptosis were observed in HL-60/neo cells. HIDAC did not affect Bcl-2 levels in either cell type. The intracellular accumulation of Ara-CTP relative to dCTP, Ara-C DNA incorporation and Ara-C-induced early DNA damage in the form of strand breaks (detected by alkaline elution assay) were not significantly different between HL-60/Bcl-2 and HL-60/neo cells. In addition, HIDAC treatment caused similar DNA synthesis inhibition in the two cell types. These results indicate that high intracellular levels of Bcl-2 operate distally to inhibit the final apototic cell death pathway by preventing the conversion of HIDAC-induced early DNA damage into lethal DNA fragmentation associated with apoptosis.

Ara-C has been shown to induce apoptosis of human acute myelogenous leukemia HL-60 cells. The DNA repair enzyme poly(ADP-ribose) polymerase (PARP) is known to be degraded during apoptosis. PARP as a substrate is cleaved by the Yama protease, encoded by the CPP32beta/Yama gene. Yama belongs to the interleukin 1beta converting enzyme/ced-3 family of cysteine proteases that are activated as a cascade, producing proteolytic cleavage of specific substrates that results in the morphological and biochemical features of apoptosis. In the present studies, we determined the effect of high intracellular levels of the antiapoptosis Bcl-2 or Bcl-xL protein on Yama protease activation and PARP degradation during Ara-C-induced apoptosis. For this, we utilized HL-60/Bcl-2, HL-60/Bcl-xL, or control HL-60/neo cells, which were created by transfection of the cDNA of the bcl-2, bcl-xL, or the neomycin-resistant genes, respectively. As compared to HL-60/neo, HL-60/Bcl-2 and HL-60/Bcl-xL cells have 5-fold greater expression of Bcl-2 and Bcl-xL, respectively. However, these cell lines have similar levels of p32Yama and PARP. Treatment with 10 or 100 microM Ara-C for 4 h produced DNA fragmentation and morphological features of apoptosis in HL-60/neo cells. This was associated with the cleavage and activation of p32Yama and PARP degradation but not with the induction of Yama mRNA. In contrast, in HL-60/Bcl-2 and HL-60/ Bcl-xL cells, Ara-C-induced p32Yama activation by its cleavage, PARP degradation and apoptosis were significantly inhibited. High Bcl-2 and Bcl-xL levels in these cells also inhibited Yama protease activity, PARP degradation, and apoptosis due to clinically relevant concentrations of etoposide and mitoxantrone. These results suggest that the activation of the Yama protease and PARP degradation are involved in Ara-C-, etoposide-, or mitoxantrone-induced apoptosis. In addition, they suggest that Bcl-2 and Bcl-xL antagonize drug-induced apoptosis by a mechanism that interferes in the activity of a key cysteine protease that is involved in the execution of apoptosis.

By suppressing apoptosis, hemopoietic growth factors (HGFs) promote the survival of CD34+, HLA-DR+ marrow cells that are enriched for hemopoietic progenitor cells (HPC). In the present studies, we have examined the effects of pIXY321, a genetically engineered fusion protein of GM-CSF and IL-3 (GM-CSF/IL-3), on high-dose Ara-C (HIDAC) and taxol-induced apoptosis and survival of a multilineage HPC, the CFU-GEMM. Exposure to 1.0 mumol/l taxol for 24 h or HIDAC > or = 10 mumol/l for 4 h induced internucleosomal DNA fragmentation and the morphologic features of apoptosis in CD34+, HLA-DR+ cells. These treatments were associated with > or = 50% inhibition of the assayable CFU-GEMM colony numbers. Incubation in serum-free medium (SFM) alone for 24 h also induced apoptosis of CD34+, HLA-DR+ cells, which was associated with reduced intracellular levels of the bcl-2 gene product p26BCL-2. Co-treatment with pIXY321 (10 ng/ml) inhibited apoptosis of CD34+, HLA-DR+ cells incubated in SFM, without significantly increasing the intracellular p26BCL-2 levels. Furthermore, co-treatment with pIXY321 significantly reduced taxol- and Ara-C-induced apoptosis and promoted the survival of CFU-GEMM (P < 0.05). Taxol and Ara-C mediated apoptosis of CD34+, HLA-DR+ cells, and its inhibition by pIXY321, was not accompanied by any significant alteration in the intracellular p26BCL-2 levels. By demonstrating that co-treatment with pIXY321 confers significant protection against apoptosis of CD34+, HLA-DR+ cells as well as promotes survival of normal HPC exposed to clinically relevant schedules and concentrations of taxol of Ara-C, these results support the design of chemotherapy regimens incorporating pIXY321 plus taxol and/or high-dose Ara-C for solid tumors and/or acute leukemias. It is hoped that the use of such a cytokine might maintain normal HPC numbers following chemotherapy, therefore avoiding prolonged suppression.

Prolonged administration of conventional (100 mg/m2/day) or low dose Ara-C (20 mg/m2/day) has been associated with significant clinical antileukemic effects in AML and myelodysplastic syndromes. These doses and schedules of Ara-C yield plasma Ara-C concentrations in the range of 10 to 100 nM. Utilizing concentrations and a schedule of Ara-C treatment, representative of Ara-C exposures in these clinical situations, we performed in vitro studies to examine the effects of co-treatment with pIXY 321 on Ara-C induced apoptosis and Ara-C-mediated colony growth inhibition of human myeloid leukemia HL-60 cells. Significantly greater internucleosomal DNA fragmentation, higher percentage of morphologically recognizable apoptotic cells and increased colony growth inhibition were observed following treatment with 100 versus 10 nM Ara-C for 5 days. Simultaneous exposure to 10 ng/ml pIXY 321 resulted in significantly increased colony growth inhibition as well as DNA fragmentation and apoptosis due to 10 nM but not 100 nM Ara-C. These concentrations of Ara-C inhibited c-myc and did not induce c-jun mRNA expression. These effects of Ara-C on c-myc and c-jun expressions were not influenced by co-treatment with pIXY 321. Neither treatment with pIXY 321 or Ara-C alone, nor co-treatment with pIXY 321 and Ara-C, significantly altered the intracellular p26BCL-2 levels in HL-60 cells. These results indicate that co-treatment with pIXY 321 significantly increases low dose Ara-C-induced apoptosis and thereby its antileukemic activity.

We investigated the ability of different doses and durations of exposure to the chemotherapeutic drugs 1-beta-D-arabinofuranosylcytosine (Ara-C), mitoxantrone (MTN), and paclitaxel (taxol, TXL) to induce internucleosomal DNA fragmentation and apoptosis in human acute myeloid leukemia (AML) HL-60 cells in suspension culture. At clinically achievable concentrations, all three drugs have been shown to induce apoptosis in HL-60 cells. An improved method was developed for the isolation of pure genomic DNA and the detection of drug-induced internucleosomal DNA fragmentation in < 1.0 microgram of DNA sample by agarose gel electrophoresis. Morphologic evidence for apoptosis was determined by light microscopy following Wright staining, and cell viability was assessed by trypan blue dye exclusion. Internucleosomal DNA fragmentation was observed following exposure to 1.0 microM Ara-C for 4 h, which increased with 10 and 50 microM Ara-C. Incubation with 100 microM Ara-C produced internucleosomal DNA fragmentation starting at 3 h, which increased with longer periods of exposure to Ara-C. Utilizing a schedule of 1-h exposure followed by 3-h suspension in drug-free medium, 0.25 microM MTN was found to initiate DNA fragmentation, which increased with exposure to 1.0 and 5.0 microM MTN. However, identical treatment with higher concentrations of MTN resulted in random DNA degradation. Alternatively, continuous exposure to 1.0 microM MTN for 3 h was necessary to initiate internucleosomal DNA fragmentation. This increased with exposure intervals of up to 6 h. Exposure to TXL concentrations as low as 0.01 microM for 24 h caused internucleosomal DNA fragmentation, which increased with dose escalation (0.05, 0.1, 0.5, and 1.0 microM) of TXL. Although continuous exposure to 1.0 microM TXL for a period as short as 8 h produced internucleosomal DNA fragmentation, this increased significantly with longer exposure intervals. In general there appears to be a threshold concentration and duration of exposure below which none of these three drugs activates endonucleolytic internucleosomal DNA fragmentation and apoptosis. This threshold is lower for the DNA-interactive drugs MTN and Ara-C but higher for the non-DNA-interactive drug TXL. Higher doses or prolonged treatments with the drugs produce random DNA fragmentation associated with necrotic cell death. These in vitro results may further improve our understanding of the antileukemic cytotoxic effects of these drugs, which may enable a more rational design of drug regimens for optimal treatment of AML.

A total of 16 patients with refractory leukemia have been entered on a phase I study employing escalating doses of a 96 h continuous infusion of high-dose cytosine arabinoside (ara-C) administered in conjunction with a 120 h infusion of a fixed dose (22 g/m2 per day) of 2'-deoxycytidine (IND 28108) as a host-protective agent. Extramedullary toxicities, even at the highest ara-C dose level (e.g. 14 g/m2 per day) were mild (e.g. CALGB grade I or II), and consisted of diarrhea, fluid retention, somnolence, hepatic dysfunction, and febrile episodes. Neutropenia and thrombocytopenia were noted in all patients, but no serious infections or bleeding episodes were encountered. Steady state plasma ara-C concentrations of 20-60 microM were observed in patients treated at the 14 g/m2 dose, and were associated with plasma ara-U concentrations approaching 1 mM. Although no complete remissions were obtained at the ara-C dose levels tested to date, three partial remissions were noted, and the large majority of patients experienced a clearing of peripheral blood blasts. In addition, several patients who failed to achieve objective responses exhibited atypically benign clinical courses following completion of therapy. These findings demonstrate that 2'-deoxycytidine protects humans from otherwise lethal doses of high-dose ara-C administered by continuous infusion, and substantially ameliorates the hematologic and non-hematologic toxicity of such regimens without completely abrogating antileukemic activity. Determination of the ultimate efficacy of this strategy will require identification of an ara-C maximum tolerated dose and evaluation of the antileukemic potential of this regimen in prospective phase 11 trials.

Recently, high dose Ara-C (HIDAC) has been shown to induce leukemic cell death in vitro by the alternative process of programmed cell death (PCD) or apoptosis which correlates with the inhibition of their clonogenic survival. Since co-treatment with hemopoietic growth facts (HGFs) GM-CSF and IL-3 have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells, we examined the effect of HGFs pIXY 321 (a GM-CSF/IL3 fusion protein) and G-CSF on HIDAC induced PCD and related gene expressions as well as HIDAC mediated colony growth inhibition of human myeloid leukemia cells. Treatment with G-CSF or pIXY 321 alone for up to 24 hours neither suppressed nor induced PCD in HL-60 or KG-1 cells. However, exposure to either of the HGFs for 20 hours followed by a combined treatment for 4 hours with HIDAC plus either of the HGFs versus HIDAC alone significantly enhanced the intracellular Ara-CTP accumulation and the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of C-jun expression but a significant repression in BCL-2 and c-myc expressions. In addition, the treatment with either of the HGFs plus HIDAC versus HIDAC alone produced a significantly greater inhibition of the clonogenic survival of the myeloid leukemia cells. These findings underscore an additional mechanism of leukemic cell death induced by HIDAC which can be modulated by the HGFs to improve the antileukemic activity of HIDAC.

High dose Ara-C (HIDAC) induces programmed cell death (PCD) or apoptosis in vitro in human myeloid leukemia cells, which correlates with the inhibition of their clonogenic survival. Hematopoietic growth factors (HGFs) granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells. We examined the effect of pIXY 321 (a GM-CSF/IL-3 fusion protein) on HIDAC-induced PCD and related gene expressions as well as HIDAC-mediated colony growth inhibition of human myeloid leukemia cells. Unlike the previously described effects of HGFs on normal bone marrow progenitor cells, exposure to pIXY 321 alone for up to 24 hours did not suppress PCD in HL-60 or KG-1 cells. However, exposure to pIXY 321 for 20 hours followed by a combined treatment with Ara-C plus pIXY 321 for 4 or 24 hours versus treatment with Ara-C alone significantly enhanced the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of c-jun expression and a significant decrease in BCL-2. In addition, the treatment with pIXY 321 plus HIDAC versus HIDAC alone produced a significantly greater inhibition of HL-60 colony growth. These findings highlight an additional mechanism of HIDAC-induced leukemic cell death that is augmented by cotreatment with pIXY 321 and may contribute toward an improved antileukemic activity of HIDAC.

The intracellular metabolism and cytotoxic effects of Ara-C and 2'-difluorodeoxycytidine (dFdC or Gemcitabine) administered with or without deoxycytidine (dCyd) were examined in cisplatin-resistant (2008/C13) and -sensitive (2008) human ovarian cystadenocarcinoma cells. Compared to 2008 cells, 2008/C13 cells possess 2.1-fold higher glutathione (GSH) levels, enhanced expressions of GSH S-transferase (GST)-pi mRNA and protein, and significantly greater activity of GST, GSH peroxidase, and GST reductase. Although 2008/C13 cells were slightly cross-resistant to 4-hydroperoxycyclophosphamide, the drug displayed a steep dose-response (colony growth inhibition) effect toward these cells. 2008/C13 cells expressed greater sensitivity toward Ara-C and Gemcitabine. This was associated with intracellular Ara-CTP and dFdCtriphosphate levels in 2008/C13 significantly higher than those in 2008 cells. Against bone marrow progenitor cells, the cytotoxic effects of submicromolar levels of Ara-C or dFdC, produced in plasma following intraperitoneal administration of the drugs, were significantly reversed by cotreatment with high levels of dCyd achieved in plasma following intravenous administration. In contrast, the metabolism and cytotoxic effects of Ara-C and dFdC in 2008 and 2008/C13 cells were not significantly altered by dCyd concentrations that are reached in the peritoneum following intravenous administration. These in vitro data suggest that systematically administered dCyd might protect bone marrow progenitor cells against Ara-C cytotoxicity without impairing antitumor activity of intraperitoneal Ara-C.

We have examined the effect of the ribonucleotide reductase inhibitors thymidine (dThd) and hydroxyurea (HU) on the metabolism and cytotoxicity of high concentrations (10 to 100 mumol/L) of cytosine arabinoside (Ara-C) in a deoxycytidine kinase-deficient, highly Ara-C-resistant human promyelocytic leukemic cell subline (HL-60/Ara-C). Administration of dThd or HU (0.5 to 3 mmol/L) in conjunction with high concentrations of Ara-C either sequentially or simultaneously lead to enhanced (and in some cases a supra-additive) inhibition of HL-60/Ara-C suspension culture growth and soft agar colony formation. In addition, exposure of cells to 0.5 mmol/L dThd or HU for 48 hours significantly increased the percentage of HL-60/Ara-C in S-phase. In contrast to previous studies involving low-dose Ara-C administration, we were unable to detect dThd- or HU-mediated increments in Ara-CTP formation in HL-60/Ara-C cells treated with high-dose Ara-C despite reductions in intracellular dCTP pools as great as 65%. However, dThd or HU did increase Ara-C incorporation into newly synthesized DNA. These studies show that administration of dThd or HU in conjunction with high concentrations of Ara-C results in an enhanced antiproliferative effect of Ara-C against Ara-C-resistant leukemic cells deficient in deoxycytidine kinase.

Hematopoietic growth factors (HGFs) interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) individually have been shown to increase the percentage of acute myeloid leukemia (AML) blasts in S phase and enhance the cytotoxic effects of Ara-C against these blasts in culture. We compared in vitro the effects of a combined treatment with GM-CSF (10 ng/mL) plus IL-3 (10 ng/mL) on the metabolism and cytotoxicity of Ara-C in normal bone marrow mononuclear cells (NBMMC) and AML blasts. NBMMC from six healthy volunteers and AML blasts from 10 patients were incubated for 20 hours with or without IL-3 plus GM-CSF, followed by a concurrent treatment with Ara-C for 4 additional hours. Exposure to the HGFs and Ara-C produced significantly higher intracellular Ara-CTP levels as well as higher Ara-CTP/dCTP pool ratios in AML blasts as compared with NBMMC. Treatment with HGFs resulted in [3H] Ara-C DNA incorporation that was significantly higher in AML blasts versus NBMMC. This selective improvement of Ara-C metabolism in AML blasts was associated with an enhanced Ara-C-mediated leukemia colony-forming unit (CFU) growth inhibition. In contrast, exposure to HGFs resulted in an improved colony growth of normal CFU granulocyte-monocyte and CFU-granulocyte, erythroid, monocyte, megakaryocyte. These in vitro studies indicate that a combined treatment with IL-3 plus GM-CSF may improve the selectivity of Ara-C against AML blasts.

We have examined the effect of recombinant interleukin 3 (rIL-3) on the metabolism of high-dose cytosine arabinoside (Ara-C), an S-phase-specific agent, in normal human bone marrow mononuclear cells (BMMC) and leukemic blasts from patients with acute myeloid leukemia (AML). Exposure to rIL-3 for 24 h significantly increased the percentage of cycling S-phase cells in normal BMMC as well as leukemic cells. A concomitant expansion of intracellular deoxycytidine triphosphate (dCTP) levels occurred to a significantly greater extent in normal BMMC. Compared to treatment with Ara-C (10 mumol/liter) alone, prior and coadministration of rIL-3 with Ara-C increased Ara-CTP levels in leukemic blasts. However, an identical treatment produced significantly higher dCTP levels in normal BMMC, resulting in a significantly lower mean Ara-CTP to dCTP pool ratio in normal BMMC compared to that observed in each of the samples of AML blasts. Following treatment with Ara-C plus rIL-3 versus Ara-C alone, the alteration in Ara-C DNA incorporation corresponded with the change in Ara-CTP to dCTP ratio observed in normal BMMC and AML blasts. The differential effect of rIL-3 on the metabolism of high-dose Ara-C in normal versus leukemic cells may indicate a role for rIL-3 in enhancing the selectivity of Ara-C toward leukemic myeloblasts.

The interaction between 2'-deoxycytidine (dCyd) and 1-beta-D-arabinofuranosylcytosine (ara-C), administered at pharmacologically achievable concentrations, was examined in four continuously cultured human leukemia cell lines, HL-60, KG-1, K-562, and CCRF-CEM. In three of the cell lines (HL-60, K-562, and CCRF-CEM), co-administration of 20 or 50 microM dCyd with 10 microM ara-C reduced ara-CTP formation by at least 90% and incorporation of ara-C into DNA by at least 80%. In contrast, KG-1 cells exhibited substantially smaller reductions in both ara-CTP formation and incorporation of ara-C into DNA under identical conditions. KG-1 cells were distinguished by the highest activity of the enzyme cytidine deaminase of the four lines assayed, and exhibited the smallest increments in the intracellular accumulation of both dCyd and deoxycytidine triphosphate (dCTP) in response to exogenous dCyd. Co-administration of 1 mM tetrahydrouridine (THU) or 0.5 mM deoxy-tetrahydrouridine (dTHU) had little effect on the ability of dCyd to antagonize ara-C metabolism in HL-60, KG-1 and K-562 cells. In contrast, these deaminase inhibitors substantially increased the intracellular accumulation of dCTP as well as the ability of dCyd to antagonize ara-CTP formation and incorporation of ara-C into DNA in KG-1 cells. THU and dTHU also permitted dCyd to antagonize ara-C growth inhibitory effects in KG-1 cells to the extent observed in the other leukemic cell lines. These studies suggest that the intracellular deamination of exogenous deoxycytidine may influence the degree to which this nucleoside antagonizes ara-C metabolism and toxicity in some leukemic cells. They also raise the possibility that deaminase inhibitors may be employed to modulate, and perhaps to improve, the therapeutic selectivity of pharmacologically relevant concentrations of ara-C and dCyd in the treatment of acute leukemia in man.

We examined the effect of a 96-h exposure to 1-beta-D arabinofuranosylcytosine (Ara-C) and deoxycytidine (dCyd) in medium lacking an exogenous source of colony-stimulating activity (phytohemagglutinin-stimulated leukocyte-conditioned medium, PHA-LCM) on the survival of normal human committed myeloid progenitor cells (day-7 and day-14 granulocyte-macrophage colony-forming units [CFU-GM] as well as leukemic progenitors (leukemic colony-forming units, L-CFU) derived from myeloblasts obtained from 13 patients with acute nonlymphocytic leukemia (ANLL). Coadministration of Ara-C (20-50 microM) in conjunction with dCyd (50-100 microM) permitted the survival of an average of 9%-57% of day-7 CFU-GM and 32%-65% day-14 CFU-GM, depending upon the relative concentrations of dCyd and Ara-C. In contrast, exposure of leukemic myeloblasts to identical regimens resulted in considerably greater reductions in L-CFU survival, which in general exceeded 90% and in some cases was 100%. In addition, exposure of leukemic myeloblasts to Ara-C and dCyd for 96 h in culture medium lacking PHA-LCM eliminated the secondary plating efficiency (PE2) of leukemic colonies in 11 of 13 samples assayed and reduced values dramatically in the remaining 2. Substantial preservation of CFU-GM formation was also noted when normal bone marrow samples depleted of T cells and marrows obtained from two patients with ANLL in remission were assayed. These studies suggest that in contrast to certain normal committed myeloid progenitor cells, leukemic progenitors, particularly those with self-renewal capacity, are highly vulnerable to a prolonged exposure to Ara-C and dCyd in the absence of an exogenous source of colony-stimulating activity. They also raise the possibility that a combined regimen utilizing chemotherapeutic agents in conjunction with modifications of long-term culture techniques may represent a novel approach to the ex vivo purging of leukemic cells from bone marrow in conjunction with autologous bone marrow transplantation.

The effect of IMPY (2,3-dihydro-1H-imidazolo[1,2-b]pyrazole) on the metabolism and cytotoxicity of subsequently administered 1-beta-D-arabinofuranosylcytosine (ara-C) was examined in the human promyelocytic leukemic cell line HL-60. Cells exposed to 3 mM IMPY for 12 hr followed by a 1-hr exposure to 1 microM [3H]ara-C accumulated 27.5 +/- 4.8 (S.D.) pmol ara-C/10(6) cells compared to 14.0 +/- 3.5 pmol/10(6) cells in untreated controls. Cells experienced greater than a 2-fold increment in 1-beta-D-arabinofuranosylcytosine 5'-triphosphate generation and retention following this same IMPY exposure and nearly a 4-fold increment in incorporation of ara-C into HL-60 nucleic acids. These alterations in ara-C metabolism were associated with a 36% reduction in the intracellular concentration of deoxycytidine 5'-triphosphate and reductions in deoxyadenosine 5'-triphosphate and deoxyguanosine 5'-triphosphate concentrations to undetectable levels. Coincubation of cells with IMPY along with other pyrimidine antagonists such as thymidine, N-(phosphonacetyl-L-aspartate), deoxyadenosine, and deoxyguanosine, produced up to 4-fold increments in ara-C intracellular accumulation. Pretreatment of HL-60 cells with 3 mM IMPY followed by a continuous exposure to 10 nM ara-C produced synergistic inhibitory effects on both suspension culture growth and soft agar clonogenicity. In contrast, exposure of normal human bone marrow progenitor cells (CFU-GM) to the same schedule of IMPY and ara-C produced subadditive or antagonistic effects on the growth of these cells in soft agar. These findings may have implications for the design of in vivo regimens using IMPY and ara-C.

A deoxycytidine kinase-deficient variant of a human promyelocytic leukemic cell line (HL-60/ara-C) has been isolated and characterized. These cells are capable of proliferating in the presence of 10(-6) M 1-beta-D-arabinofuranosylcytosine (ara-C), a level achieved in the plasma of leukemic patients undergoing conventional-dose ara-C therapy. The cells share numerous biological and biochemical features with the parent line, including: morphology; rate of growth; cloning characteristics; karyotype; rates of DNA, RNA, and protein synthesis; and ability to undergo terminal differentiation in the presence of agents such as 12-O-tetradecanoylphorbol acetate and dimethyl sulfoxide. In contrast, these cells display a great reduction in the total intracellular accumulation of ara-C following a 4-hr exposure to 10(-6) M ara-C (2.4 versus 99.0 pmol ara-C/10(6) cells). Resistant cells exposed to 10(-6) M ara-C for 1 hr also exhibited a reduction in the generation [1.2 versus 31.9 pmol 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP)/10(6) cells] and the 4-hr retention (0.30 versus 3.87 pmol ara-CTP/10(6) cells) of ara-CTP, the lethal ara-C metabolite, in comparison to parent cells. Incorporation of ara-C into resistant HL-60 cell DNA was also profoundly decreased. These biochemical alterations were associated with a 1000-fold decrease in the sensitivity of clonogenic cells to continuously administered ara-C (ara-C 50% inhibitory concentration: 1.8 X 10(-6) M for HL-60/ara-C; 3.0 X 10(-9) M for HL-60). A variety of antagonists of de novo pyrimidine synthesis inhibited the growth of ara-C-sensitive and -resistant cells to a similar extent. When HL-60 cells were exposed to a lethal concentration of thymidine (5 X 10(-3) M), coadministration of 5 X 10(-6) M deoxycytidine restored 90 +/- 4% (S.D.) of colony-forming capacity. Normal human bone marrow progenitor cells were protected to a similar degree by 3 X 10(-3) M deoxycytidine. In contrast, deoxycytidine concentrations as high as 5 X 10(-3) M were unable to confer any protection to HL-60/ara-C cells under identical conditions. These studies suggest that an enzymatic perturbation rendering human leukemic cells highly resistant to ara-C may be exploited to achieve a selective in vitro chemotherapeutic effect.

We examined the ability of high concentrations of the naturally occurring nucleoside deoxycytidine (dCyd) to reverse the cytotoxicity of high (eg, greater than or equal to 10(-5) mol/L) concentrations of 1-B-D arabinofuranosylcytosine (Ara-C) toward normal (CFU-GM) and leukemic myeloid progenitor cells (L-CFU). Leukemic myeloblasts from patients with acute nonlymphocytic leukemia (ANLL) and normal human bone marrow mononuclear cells were cultured in soft agar in the continuous presence of 10(-5) to 5 X 10(-5) mol/L of Ara-C together with dCyd (10(-4) to 5 X 10(-3) mol/L). Administration of 10(-5) mol/L of Ara-C alone eradicated colony formation in all samples tested. Coadministration of 10(-3) mol/L of dCyd restored 72.2% of control colony formation for CFU-GM, but only 10.9% for L-CFU. When higher concentrations of Ara-C (eg, 5 X 10(-5) mol/L) were administered, dCyd-mediated protection toward CFU-GM decreased, but remained significantly greater than that observed for L-CFU. Incubation with 10(-3) mol/L of dCyd reduced the 4-hour intracellular accumulation of the triphosphate derivative of Ara-C (Ara-CTP) in both normal and leukemic cells by greater than 98%; under identical conditions, a significant expansion of the intracellular of the triphosphate derivative of dCyd (dCTP) pools was observed in normal bone marrow mononuclear cells but not in leukemic blasts. This finding was associated with a greater reduction in Ara-C DNA incorporation in normal elements. These in vitro studies suggest that dCyd may preferentially protect normal v leukemic myeloid progenitor cells from the lethal actions of high-dose Ara-C.

Human recombinant GM-CSF (rGM-CSF) is under investigation as a growth-protective agent for normal hematopoietic elements in phase I trials of myelosuppressive chemotherapy and in bone marrow transplantation. We determined the effect of rGM-CSF on the metabolism of high dose Ara-C in bone marrow mononuclear cells (BMMCs) from healthy volunteers and patients with ANLL. Cells were incubated with rGM-CSF alone, Ara-C alone, or a combination of the two drugs. Treatment with rGM-CSF alone yielded approximately a twofold increment in intracellular dCTP pools in normal BMMCs but not in leukemic blasts. Exposure to rGM-CSF in conjunction with Ara-C corrected Ara-C-mediated declines in dCTP levels and decreased cytosine arabinoside triphosphate (Ara-CTP) accumulation in normal BMMCs but not in their leukemic counterparts. Furthermore, when exposure to Ara-C was preceded by treatment with rGM-CSF for 18 hr, an even greater reduction in the Ara-CTP/dCTP pool ratio was observed in normal versus leukemic elements; however, this did not significantly change Ara-C DNA incorporation in the two cell types. The differential effect of rGM-CSF on the phosphorylation of Ara-C in normal BMMCs versus leukemic blasts has potential implications for the use of a regimen consisting of rGM-CSF and high dose Ara-C in the treatment of ANLL with chemotherapy or autologous bone marrow transplantation.