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Although bone marrow mesenchymal stem cells (BM-MSCs)-derived exosomes have been reported to be closely associated with acute myeloid leukemia (AML) progression and chemo-resistance, but its detailed functions and molecular mechanisms have not been fully delineated. Besides, serum RNA m6A demethylase fat mass and obesity-associated protein (FTO)-containing exosomes are deemed as important indicators for cancer progression, and this study aimed to investigate the role of BM-MSCs-derived FTO-exosomes in regulating the malignant phenotypes of AML cells. Here, we verified that BM-MSCs-derived exosomes delivered FTO to promote cancer aggressiveness, stem cell properties and Cytosine arabinoside (Ara-C)-chemoresistance in AML cells, and the underlying mechanisms were also uncovered. Our data suggested that BM-MSCs-derived FTO-exo demethylated m6A modifications in the m6A-modified LncRNA GLCC1 to facilitate its combination with the RNA-binding protein Hu antigen R (HuR), which further increased the stability and expression levels of LncRNA GLCC1. In addition, LncRNA GLCC1 was verified as an oncogene to facilitate cell proliferation and enhanced Ara-C-chemoresistance in AML cells. Further experiments confirmed that demethylated LncRNA GLCC1 served as scaffold to facilitate the formation of the IGF2 mRNA binding protein 1 (IGF2BP1)-c-Myc complex, which led to the activation of the downstream tumor-promoting c-Myc-associated signal pathways. Moreover, our rescuing experiments validated that the promoting effects of BM-MSCs-derived FTO-exo on cancer aggressiveness and drug resistance in AML cells were abrogated by silencing LncRNA GLCC1 and c-Myc. Thus, the present firstly investigated the functions and underlying mechanisms by which BM-MSCs-derived FTO-exo enhanced cancer aggressiveness and chemo-resistance in AML by modulating the LncRNA GLCC1-IGF2BP1-c-Myc signal pathway, and our work provided novel biomarkers for the diagnosis, treatment and therapy of AML in clinic., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cytarabine (Ara-C) is widely used in the induction chemotherapy for acute myeloid leukemia (AML). Association between LncRNA GAS5 genetic polymorphism and the recovery of hematopoietic function after Ara-C-based chemotherapy is observed. This study aimed to identify whether intervention of GAS5 expression and GAS5 genotype affect Ara-C-induced inhibition of hematopoietic stem cells (HSCs) differentiation. In this study, cord blood-derived CD34 + cells were cultured in vitro, and a cell model of myelosuppression was established by treatment of CD34 + cells with Ara-C. The effect of GAS5 overexpression, Ara-C treatment, and GAS5 rs55829688 genotype on the hematopoietic colony-forming ability of CD34 + cells was assessed using methylcellulose-based colony forming unit assay. GAS5 overexpression slowed down the proliferation of cord blood-derived CD34 + cells significantly (p < 0.05) and decreased their ability to form hematopoietic colonies in vitro. Ara-C significantly reduced the hematopoietic colony-forming ability of CD34 + cells in vitro (p < 0.0001), and overexpressing GAS5 further decreased the number of hematopoietic colonies. GAS5 expression was higher in CD34 + cells than in CD34 - cells, and positively correlated with GATA1 mRNA expression in CD34 + cells in vitro culture. However, GAS5 genotype had no effect on the total number of hematopoietic colonies formed from cord blood-derived CD34 + cells. In conclusion, our study highlights that GAS5 inhibited the in vitro proliferation and reduced the hematopoietic colony-forming ability of cord blood-derived CD34 + cells, with the most pronounced effect observed on CFU-GEMM formation. GAS5 also enhanced the inhibitory effect of Ara-C on the in vitro hematopoietic ability of CD34 + HSCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Acute myeloid leukaemia (AML) is very common haematopoietic malignancies with poor prognosis. Chemotherapy is still a mainstay therapy for AML patients. AML microenvironment plays critical roles in therapy response. However, the role of chemotherapy in AML microenvironment is poorly understood. In this study, we report that cytarabine (AraC)-triggered TNFα from AML cells expanded myeloid-derived suppressor cells (MDSCs) and enhanced MDSC functions and survival through activating IL-6/STAT3 and NFκB pathways. Blockade of TNFα in conditioned medium-derived AraC-treated AML cells (AraC&#95;CM) impaired MDSC expansion and functions, reduced IL-6 secretion and the level of activated STAT3. Inhibiting IL6 or STAT3 abrogated AraC&#95;CM-mediated MDSC suppressive function. Additionally, inhibiting TNFα also impaired AraC&#95;CM-mediated NFκB activation. Blocking NFκB activation reduced MDSC viability induced by AraC&#95;CM. Together, these results provided a role of AraC-induced TNFα in MDSC expansion and functions and suggest that targeting TNFα may benefit AML patients to current anticancer strategies by blocking MDSC-mediated immunosuppression., (© 2022 The Scandinavian Foundation for Immunology.)

Acute myeloid leukemia (AML) is a hematological malignancy with a high risk of relapse. This issue is associated with the development of mechanisms leading to drug resistance that are not yet fully understood. In this context, we previously showed the clinical significance of the ATP binding cassette subfamily B-member 1 (ABCB1) in AML patients, namely its association with stemness markers and an overall worth prognosis. Calcium signaling dysregulations affect numerous cellular functions and are associated with the development of the hallmarks of cancer. However, in AML, calcium-dependent signaling pathways remain poorly investigated. With this study, we show the involvement of the ORAI1 calcium channel in store-operated calcium entry (SOCE), the main calcium entry pathway in non-excitable cells, in two representative human AML cell lines (KG1 and U937) and in primary cells isolated from patients. Moreover, our data suggest that in these models, SOCE varies according to the differentiation status, ABCB1 activity level and leukemic stem cell (LSC) proportion. Finally, we present evidence that ORAI1 expression and SOCE amplitude are modulated during the establishment of an apoptosis resistance phenotype elicited by the chemotherapeutic drug Ara-C. Our results therefore suggest ORAI1/SOCE as potential markers of AML progression and drug resistance apparition.

Acute myeloid leukemia (AML) accounts for around 20% of diagnosed childhood leukemia. Cytarabine (CYT) is involved in the AML treatment regimen. AML and CYT showed impairment in spermatogenesis in human and rodents in adulthood. We successfully developed an AML disease model in sexually immature mice. Monocytes and granulocytes were examined in all groups: untreated control, AML alone, CYT alone and AML+CYT (in combination). There was a significant increase in the counts of monocytes and granulocytes in the AML-treated immature mice (AML) compared to the control, and AML cells were demonstrated in the blood vessels of the testes. AML alone and CYT alone impaired the development of spermatogenesis at the adult age of the AML-treated immature mice. The damage was clear in the structure/histology of their seminiferous tubules, and an increase in the apoptotic cells of the seminiferous tubules was demonstrated. Our results demonstrated a significant decrease in the meiotic/post-meiotic cells compared to the control. However, CYT alone (but not AML) significantly increased the count of spermatogonial cells (premeiotic cells) that positively stained with SALL4 and PLZF per tubule compared to the control. Furthermore, AML significantly increased the count of proliferating spermatogonial cells that positively stained with PCNA in the seminiferous tubules compared to the control, whereas CYT significantly decreased the count compared to the control. Our result showed that AML and CYT affected the microenvironment/niche of the germ cells. AML significantly decreased the levels growth factors, such as SCF, GDNF and MCSF) compared to control, whereas CYT significantly increased the levels of MCSF and GDNF compared to control. In addition, AML significantly increased the RNA expression levels of testicular IL-6 (a proinflammatory cytokine), whereas CYT significantly decreased testicular IL-6 levels compared to the control group. Furthermore, AML alone and CYT alone significantly decreased RNA expression levels of testicular IL-10 (anti-inflammatory cytokine) compared to the control group. Our results demonstrate that pediatric AML disease with or without CYT treatment impairs spermatogenesis at adult age (the impairment was more pronounced in AML+CYT) compared to control. Thus, we suggest that special care should be considered for children with AML who are treated with a CYT regimen regarding their future fertility at adult age.

Aberrant activation of the hedgehog (HH) pathway is observed in many neoplasms, including acute myeloid leukemia (AML). The glioma-associated oncogene homolog (GLI) transcription factors are the main downstream effectors of the HH signaling cascade and are responsible for the proliferation and maintenance of leukemic stem cells, which support chemotherapy resistance and leukemia relapse. Cytarabine (Ara-C)-resistant variants of AML cell lines were established through long-term cultivation with successively increasing Ara-C concentrations. Subsequently, differences in GLI expression were analyzed by RT-qPCR. GLI3 mRNA levels were detectable in parental Kasumi-1, OCI-AML3, and OCI-AML5 cells, whereas GLI3 expression was completely silenced in all resistant counterparts. Therefore, we generated GLI3-knockdown cell lines using small hairpin RNAs (shRNA) and evaluated their sensitivity to Ara-C in vitro. The knockdown of GLI3 partly abolished the effect of Ara-C on colony formation and induction of apoptosis, indicating that GLI3 downregulation results in Ara-C resistance. Moreover, we analyzed the expression of several genes involved in Ara-C metabolism and transport. Knockdown of GLI3 resulted in the upregulation of SAM and HD domain-containing protein 1 ( SAMHD1 ), cytidine deaminase ( CDA ), and ATP-binding cassette C11 ( ABCC11 )/multidrug resistance-associated protein 8 ( MRP8 ), each of which has been identified as a predictive marker for Ara-C response in acute myeloid leukemia. Our results demonstrate that GLI3 downregulation is a potential mechanism to induce chemotherapy resistance in AML.

Introduction: Acute myeloid leukaemia is the most common type of acute leukaemia in the world. Thus, the study of genetic alterations, such as single-nucleotide polymorphisms (SNPs), has contributed to a better understanding of the mechanisms underlying leukaemogenesis, to improve the prognosis and to increase the survival of these patients. However, there is no synthesis of evidence in the literature evaluating the quality of evidence and the risk of bias in the studies such that the results can be translated. Thus, this systematic review protocol aims to assess the impact of SNPs on genes involved in the metabolism of cytarabine and anthracyclines with respect to survival, treatment response and toxicity in patients with AML., Methods: This systematic review protocol is based on PRISMA guidelines and includes searches in six electronic databases, contact with authors, repositories of clinical trials, and cancer research. Studies published in peer-reviewed journals will be included if they meet the eligibility criteria: (a) samples composed of individuals of any age, of both sexes, with a diagnosis of AML, regardless of the time of diagnosis of disease; (b) participants who have undergone or are undergoing cytarabine- and anthracycline-associated chemotherapy or cytarabine-only chemotherapy; and (c) in vivo studies. Studies that include patients with promyelocytic leukaemia (Fab type 3) will be excluded because this disease has different treatment. The process of study selection, data extraction, and evaluation/synthesis will be performed in duplicate. Assessment of methodological quality and risk of bias will be performed using the Cochrane Risk of Bias Tool for randomized clinical studies and the Downs-Black Checklist for cohort and case-control studies. The synthesis of evidence will include the level of evidence based on the GRADE protocol. A meta-analysis of the association between SNPs and outcomes may be performed based on Cochrane guidelines., Discussion: It is expected that clinical decisions for AML patients will consider evidence-based practices to contribute to better patient management. In this way, we will be able to define how to treat patients with AML to improve their survival and quality of life., Systematic Review Registration: PROSPERO CRD42018100750.

Aim: Cytarabine (Ara-C), a mainstay of acute myeloid leukemia (AML) treatment, is a prodrug requiring activation to ara-CTP for its antileukemic activity. Aim of this study was to evaluate impact of genetic variants in the key genes involved in ara-C metabolism on the leukemic cell intracellular levels of ara-CTP., Method: We investigated SNPs in 14 ara-C metabolic-pathway genes, for association with intracellular ara-CTP levels, in leukemic cells obtained post-initiation of cytarabine infusion in pediatric AML patients (n = 68)., Results: Nine SNPs were significantly associated with leukemic cell intracellular concentration of ara-CTP. A comprehensive ara-CTP-SNP-score (ACSS) was further developed from top four SNPs identified in regression model. Patients were classified into three groups based on ACSS: high-ACSS (score >0), intermediate-ACSS (score = 0) and low-ACSS (score <0). ACSS designation was significant predictor of intracellular ara-CTP levels (p = 0.00012), suggesting a cumulative or synergistic effect of the significant SNPs., Conclusion: ACSS score designation holds promise in definfing ara-C dose. Validation of the clinical utility of ACSS score in other independent cohorts will help identification of patients with potentially lower or higher levels of the ara-CTP in leukemic cells, thereby opening up opportunities for dose management to reduce toxicity and enhance efficacy.

Background: Cytarabine arabinoside (Ara-C) has been the core of chemotherapy for adult acute myeloid leukemia (AML). Ara-C undergoes a three-step phosphorylation into the active metabolite Ara-C triphosphosphate (ara-CTP). Several enzymes are involved directly or indirectly in either the formation or detoxification of ara-CTP., Methods: A total of 12 eQTL (expression Quantitative Trait Loci) single nucleotide polymorphisms (SNPs) or tag SNPs in 7 genes including CMPK1, NME1, NME2, RRM1, RRM2, SAMHD1 and E2F1 were genotyped in 361 Chinese non-M3 AML patients by using the Sequenom Massarray system. Association of the SNPs with complete remission (CR) rate after Ara-C based induction therapy, relapse-free survival (RFS) and overall survival (OS) were analyzed., Results: Three SNPs were observed to be associated increased risk of chemoresistance indicated by CR rate (NME2 rs3744660, E2F1 rs3213150, and RRM2 rs1130609), among which two (rs3744660 and rs1130609) were eQTL. Combined genotypes based on E2F1 rs3213150 and RRM2 rs1130609 polymorphisms further increased the risk of non-CR. The SAMHD1 eQTL polymorphism rs6102991 showed decreased risk of non-CR marginally (P = 0.055). Three SNPs (NME1 rs3760468 and rs2302254, and NME2 rs3744660) were associated with worse RFS, and the RRM2 rs1130609 polymorphism was marginally associated with worse RFS (P = 0.085) and OS (P = 0.080). Three SNPs (NME1 rs3760468, NME2 rs3744660, and RRM1 rs183484) were associated with worse OS in AML patients., Conclusion: Data from our study demonstrated that SNPs in Ara-C and dNTP metabolic pathway predict chemosensitivity and prognosis of AML patients in China.

Background: Gene polymorphisms at microRNA-binding sites (poly-miRTS) may affect gene transcription and expression through miRNA regulation, which is associated with cancer susceptibility, sensitivity to chemotherapy and prognosis. This study investigated the association between poly-miRTS of Ara-C/anthracycline metabolic pathways genes and the outcome of acute myeloid leukemia (AML) in Chinese patients after Ara-C-based chemotherapy., Methods: A total of 17 poly-miRTS were selected from the SNPinfo Web Server and genotyped in 206 Chinese Han non-FAB-M3 AML patients using the SEQUENOM Mass-ARRAY system., Results: Among these 17 poly-miRTS, five Ara-C metabolic gene single nucleotide polymorphisms (SNPs, NT5C2 rs10786736 and rs8139, SLC29A1 rs3734703, DCTD rs7278, and RRM1 rs1042919) were identified to significantly associate with complete AML remission and/or overall and relapse-free survival (OS and RFS, respectively), and four anthracycline-metabolic gene SNPs (ABCC1 rs3743527, rs212091, and rs212090 and CBR1 rs9024) were significantly associated with chemotherapy-related toxicities. Moreover, SLC29A1 rs3734703 was shown to associate with both chemotherapy response and survival (adjusted OR 2.561 in the overdominant model; adjusted HR 2.876 for OS and 2.357 for RFS in the dominant model)., Conclusions: The data from the current study demonstrated that the poly-miRTS of Ara-C/anthracyclines metabolic genes predicted the sensitivity and side effects of AML to Ara-C-based chemotherapy and patient survival. Further study will confirm them as biomarkers for AML patients after Ara-C-based chemotherapy.

Arabinosylcytosine (araC) is a mainstay in the initial treatment of acute myeloid leukemia (AML), although relapses are common. Given the recent recognition of altered DNA methylation patterns in relapsed AML, we considered whether araC, which acts by incorporation into DNA, could itself perturb methylation dynamics. To explore this possibility, we examined several DNA methyltransferases and find that araC embedded in DNA is consistently methylated with an efficiency diminished relative to that of deoxycytidine. Importantly, with the human maintenance methyltransferase DNMT1, the extent of araC methylation is reduced by more than ∼200-fold. These observations support a model whereby araC treatment may itself contribute to locus-specific, passive DNA demethylation in relapsed AML.

Background: Variation in terms of outcome and toxic side effects of treatment exists among acute myeloid leukemia (AML) patients on chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Candidate Ara-C metabolizing gene expression in primary AML cells is proposed to account for this variation., Methods: Ex vivo Ara-C sensitivity was determined in primary AML samples using MTT assay. mRNA expression of candidate Ara-C metabolizing genes were evaluated by RQPCR analysis. Global gene expression profiling was carried out for identifying differentially expressed genes between exvivo Ara-C sensitive and resistant samples., Results: Wide interindividual variations in ex vivo Ara-C cytotoxicity were observed among samples from patients with AML and were stratified into sensitive, intermediately sensitive and resistant, based on IC50 values obtained by MTT assay. RNA expression of deoxycytidine kinase (DCK), human equilibrative nucleoside transporter-1 (ENT1) and ribonucleotide reductase M1 (RRM1) were significantly higher and cytidine deaminase (CDA) was significantly lower in ex vivo Ara-C sensitive samples. Higher DCK and RRM1 expression in AML patient's blast correlated with better DFS. Ara-C resistance index (RI), a mathematically derived quotient was proposed based on candidate gene expression pattern. Ara-C ex vivo sensitive samples were found to have significantly lower RI compared with resistant as well as samples from patients presenting with relapse. Patients with low RI supposedly highly sensitive to Ara-C were found to have higher incidence of induction death (p = 0.002; RR: 4.35 [95% CI: 1.69-11.22]). Global gene expression profiling undertaken to find out additional contributors of Ara-C resistance identified many apoptosis as well as metabolic pathway genes to be differentially expressed between Ara-C resistant and sensitive samples., Conclusion: This study highlights the importance of evaluating expression of candidate Ara-C metabolizing genes in predicting ex vivo drug response as well as treatment outcome. RI could be a predictor of ex vivo Ara-C response irrespective of cytogenetic and molecular risk groups and a potential biomarker for AML treatment outcome and toxicity. Original submitted 22 December 2014; Revision submitted 9 April 2015.

Sodium metaarsenite (NaAs2O3: code name KML001) is an orally bioavailable arsenic compound with potential anti-cancer activity. However, the effect of KML001 has not been studied in acute myeloid leukemia (AML). We investigated the anti-leukemic effect of KML001 in AML, and determined the mode of action of KML001. KML001 inhibited the cellular proliferation in all AML cell lines and primary AML blasts as well as HL-60R (cytosine arabinoside-resistant HL-60) cells, while As2O3 was not effective in primary AML blasts and AML cell lines including HL-60R cells. KML001 induced G1 arrest and apoptosis in HL-60 and HL-60R cells. KML001 inhibited the activation of STAT (signal transducer and activator of transcription) 1, 3, 5, NF-κB, AKT and PI3K, while phosphorylated PTEN was upregulated. In addition, activation of ERK, p38 and JNK was observed in KML001-induced growth inhibition of HL-60 and HL-60R cells. Furthermore, KML001 induced telomeric terminal restriction fragment (TRF) length shortening in a time-dependent manner in HL-60 and HL-60R cells. Real‑time PCR with RNA extracted from KML001-treated HL-60 and HL-60R cells showed a significant reduction of catalytic subunit of telomerase, hTERT, in a time-dependent manner. Additionally, γ-H2AX, a sensitive molecular marker of DNA damage, in HL-60 and HL-60R cells was induced by KML001. These results suggest that KML001 inhibits the proliferation of AML cells including cytosine arabinoside-resistant AML cells via various mechanisms such as cell cycle arrest, induction of apoptosis, inhibition of JAK/STAT and PI3K pathways, activation of MAPK pathway and telomere targeting.

Drug resistance is a major hurdle in oncology. Responses of acute myeloid leukaemia (AML) patients to cytarabine (Ara-C)-based therapies are often short lived with a median overall survival of months. Therapies are under development to improve outcomes and include targeting the eukaryotic translation initiation factor (eIF4E) with its inhibitor ribavirin. In a Phase II clinical trial in poor prognosis AML, ribavirin monotherapy yielded promising responses including remissions; however, all patients relapsed. Here we identify a novel form of drug resistance to ribavirin and Ara-C. We observe that the sonic hedgehog transcription factor glioma-associated protein 1 (GLI1) and the UDP glucuronosyltransferase (UGT1A) family of enzymes are elevated in resistant cells. UGT1As add glucuronic acid to many drugs, modifying their activity in diverse tissues. GLI1 alone is sufficient to drive UGT1A-dependent glucuronidation of ribavirin and Ara-C, and thus drug resistance. Resistance is overcome by genetic or pharmacological inhibition of GLI1, revealing a potential strategy to overcome drug resistance in some patients.

Cytarabine (ara-C) is the key agent for treating acute myeloid leukemia (AML). After being transported into leukemic cells by human equilibrative nucleoside transporter 1 (hENT1), ara-C is phosphorylated to ara-C triphosphate (ara-CTP), an active metabolite, and then incorporated into DNA, thereby inhibiting DNA synthesis. Deoxycytidine kinase (dCK) and cytosolic 5'-nucleotidase II (cN-II) are associated with the production of ara-CTP. Because ara-C's cytotoxicity depends on ara-CTP production, parameters that are most related to ara-CTP formation would predict ara-C sensitivity and the clinical outcome of ara-C therapy. The present study focused on finding any correlation between the capacity to produce ara-CTP and ara-C-metabolizing factors. In vitro ara-CTP production, mRNA levels of hENT1, dCK, and cN-II, and ara-C sensitivity were evaluated in 34 blast samples from 33 leukemic patients including 26 with AML. A large degree of heterogeneity was seen in the capacity to produce ara-CTP and in mRNA levels of hENT1, dCK, and cN-II. Despite the lack of any association between each of the transcript levels and ara-CTP production, the ratio of dCK/cN-II transcript levels correlated significantly with the amount of ara-CTP among AML samples. The HL-60 cultured leukemia cell line and its three ara-C-resistant variants (HL-60/R1, HL-60/R2, HL-60/R3), which were 8-, 10-, and 500-fold more resistant than HL-60, respectively, were evaluated similarly. The dCK/cN-II ratio was again proportional to ara-CTP production and to ara-C sensitivity. The dCK/cN-II ratio may thus predict the capacity for ara-CTP production and ultimately, ara-C sensitivity in AML.

Purpose: Successful treatment of acute myeloid leukemia (AML) remains a therapeutic challenge, with a high percentage of patients suffering from persistent or relapsed disease. Resistance to drug therapy can develop from increased drug export and/or altered intracellular signaling. Both mechanisms are mediated by the efflux transporters ABCC4 (MRP4), ABCC5 (MRP5), and ABCC11 (MRP8), which are involved in cellular efflux of endogenous signaling molecules (e.g., cyclic adenosine 3', 5'-monophosphate and cyclic guanosine 3',5'-monophosphate) and nucleoside analogues. The nucleoside analogue cytosine arabinoside (AraC) is administered to all patients with AML., Experimental Design: Expression of ABCC transporters MRP4, MRP5, and MRP8 in blast samples from 50 AML patients was investigated by real-time reverse transcription-PCR analysis and correlated with clinical outcome measures. Accumulation of radiolabeled AraC, transport of AraC metabolites, and AraC cytotoxicity were analyzed in MRP8-transfected LLC-PK1 cells., Results: Regression analysis revealed that high expression of MRP8 is associated with a low probability of overall survival assessed over 4 years (P<0.03). MRP8-transfected LLC-PK1 cells accumulated reduced intracellular levels of AraC (63% of the parental vector-transfected LLC-PK1 control cells) as well as AraC metabolites. Furthermore, AraC monophosphate was transported by MRP8-enriched membrane vesicles (116+/-6 versus 65+/-13 pmol/mg/10 minutes by control vesicles), and MRP8-transfected cells were resistant to AraC., Conclusion: These data suggest that MRP8 is differentially expressed in AML blasts, that expression of MRP8 serves as a predictive marker for treatment outcome in AML, and that efflux of AraC metabolites by MRP8 is a mechanism that contributes to resistance of AML blasts.

Quiescent cells pose a formidable challenge in clinical management of leukemia, since they escape chemo-radiotherapy and become a source of post-therapy relapse. These cells may be refractory to various known growth-promoting signals, making it imperative to identify the biochemical signals necessary to coax them into mitosis. Using serum-starved cell lines as an experimental model of quiescent leukemic cells (QLCs), we demonstrate that a suppression of p38 stress kinase by pharmacological means forms a sufficient trigger to induce proliferative responses in the treated QLCs, even in the absence of any external growth-promoting stimulus. A robust expression of Ki67 and B23 was seen in treated cells, an effect clearly mediated through the activation of extra-cellular signal-regulated kinase (MEK/ERK) pathway. Commensurate with their proliferative status, the treated QLCs got sensitized to significantly low concentrations of anti-mitotic agents. Most importantly, primitive leukemic progenitors present in the mononuclear cells (MNCs) that were isolated from the peripheral blood of freshly diagnosed untreated acute myeloid leukemic (AML) patients got more efficiently killed by cytosine arabinoside (AraC), when the cells were pre-treated with a pharmacological inhibitor of p38. Our data strongly suggest that a suppression of p38 leads to the sensitization of QLCs to anti-mitotic drugs by triggering proliferative responses in them. This approach may have a potential clinical application.

Acute myelogenous leukemia (AML) is the most common form of acute leukemia in adults. After diagnosis, patients with AML are mainly treated with standard induction chemotherapy combining cytarabine (araC) and anthracyclines. The majority of them achieve complete remission (CR) (65-80%). However, prospects for long-term survival are poor for the majority of patients. Resistance to chemotherapy therefore remains a major obstacle in the effective treatment of patients with AML. In this review, we highlight the current knowledge of substrate cycles involved in normal deoxynucleoside triphosphate (dNTPs) metabolism and their possible role in drug resistance to araC.

A major problem in the treatment of leukemia is the development of resistance to chemotherapeutic agents. Assessing the drug resistance of leukemic cells is therefore an important aspect of treatment. One of the main mechanisms of resistance is rapid drug efflux mediated by various members of the ATP-binding cassette transporter superfamily, such as multidrug resistance gene 1 (MDR1), which encodes P-glycoprotein, multidrug resistance-associated protein (MRP) 1 and lung resistance protein. To quantify the degree of acquisition of resistance, several techniques, including drug-sensitivity studies, flow cytometry assay and quantitative gene analysis, have been developed to detect MDR1 and MRP1 gene expression in leukemic cells. However, a significant number of patients may relapse in spite of low expression of MDR1 or MRP1, suggesting the involvement of other intracellular mechanisms, possibly related to cytarabine resistance. This review focuses on the methods aimed at the assessment of drug resistance in acute myeloid leukemia.

The mechanism of 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) action was investigated in human lymphoblastoid CEM cells and myeloblastic leukemia ML-1 cells. CNDAC was metabolized to its 5'-triphosphate and incorporated into DNA, which was associated with inhibition of DNA synthesis. After incubation of cells with [(3)H]CNDAC, metabolites were detected in 3'-->5' phosphodiester linkage and at the 3' terminus of cellular DNA. Specific enzymatic hydrolysis of DNA demonstrated that the parent nucleoside and its 2'-epimer 2'-C-cyano-2'-deoxy-2-ribo-pentofuranosylcytosine accounted for approximately 65% of the total analogs incorporated into DNA and essentially all of the drug in the 3'-->5' phosphodiester linkage. In contrast, all detectable radioactivity at 3' termini was associated with 2'-C-cyano-2',3'-didehydro-2',3'-dideoxycytidine. This de facto DNA chain-terminating nucleotide arises from an electronic characteristic and cleavage of the 3'-phosphodiester bond subsequent to the addition of a nucleotide to the incorporated CNDAC moiety by beta-elimination, a process that generates a single strand break in DNA. Investigation of the biological consequences of these actions indicated that, after incubation with cytostatic concentrations of CNDAC, cell cycle progression was delayed during S phase, but that cells arrested predominantly in the G(2) phase. This differed from the S phase-arresting actions of ara-C and gemcitabine, other deoxycytidine analogs that inhibit DNA replication but do not cause strand breaks. Thus, once incorporated into DNA, the CNDAC molecule appears to act by a dual mechanism that 1) delays the progress of further DNA replication, but 2) upon addition of a deoxynucleotide results in the conversion of the incorporated analog to a de facto DNA chain terminator at the 3' terminus of a single strand break. It is likely that DNA strand breaks trigger cell cycle arrest in G(2).

Arabinosylcytosine (ara-C) is the most effective nucleoside analogue for treatment of acute myelogenous leukemia. The cytotoxicity of ara-C depends on its conversion to the triphosphate ara-CTP. In plasma, a major metabolite of ara-C is its deamination product, arabinosyluracil (ara-U). Both ara-U and ara-U monophosphate have been detected in primary leukemia cells during in vitro investigations. Because other ara-U metabolites, especially the triphosphate (ara-UTP), may serve as additional effectors of cytotoxicity, the present study investigated whether ara-UTP accumulates in circulating leukemia blasts during ara-C therapy. Patients with relapsed acute myelogenous leukemia received 2- or 4-h infusions of 0.5 g/m2/h ara-C. Intracellular accumulation of ara-CTP and ara-UTP in circulating leukemia blasts from six patients was quantitated by high-pressure liquid chromatography, revealing that ara-UTP accumulated during ara-C therapy. The intracellular concentration of ara-UTP ranged from 6-50 microM and was between 2 and 10% of the accumulated ara-CTP. In circulating blasts, ara-UTP was maintained for several hours after the end of ara-C infusion. Leukemia blasts from patients (n=27) were incubated for 1-2 h with 1, 10, or 25 microM [3H]ara-C, and radiolabeled metabolites of ara-C were separated and quantitated by high-pressure liquid chromatography. Consistent with data obtained during ara-C therapy, [3H]ara-UTP also accumulated in blasts from all these patients during in vitro incubations with [3H]ara-C. The concentration of ara-UTP after 1 h of incubation ranged from 0.2-40 microM. Incubation of cells with the cytidine deaminase inhibitor tetrahydrouridine did not perturb ara-UTP accumulation, whereas incubation with the deoxycytidylate deaminase inhibitor tetrahydrodeoxyuridine suppressed ara-UTP formation from ara-C. These observations suggested that ara-UTP is generated through deamination of ara-C monophosphate to ara-U monophosphate by deoxycytidylate deaminase, followed by its phosphorylation to ara-UTP. Consistent with these results, incubation of blasts with up to 100 microM [3H]ara-U did not result in ara-UTP accumulation, indicating that ara-U is not phosphorylated directly in these cells. The present study demonstrated that circulating leukemia blasts accumulate ara-UTP during in vitro incubations with ara-C and during ara-C therapy.

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.

The current study was undertaken to determine the relevance of leukemic blast cell proliferative activity, cellular parameters of Ara-C metabolism and the in vitro sensitivity to GM-CSF in association with the clinical response to TAD-9 induction therapy in 66 patients with de novo acute myeloid leukemia (AML). Proliferative activity was assessed by 3H-thymidine (3H-TdR) incorporation and thymidine kinase (TK) activity, parameters of Ara-C metabolism comprised the activities of deoxycytidine kinase (DCK) and DNA polymerase alpha (poly alpha) as well as Ara-CTP concentrations and 3H-Ara-C uptake into DNA. GM-CSF sensitivity was determined by in vitro incubation of blasts for 48 h with or without GM-CSF (100 U/ml) followed by an additional 4 h concurrent exposure to GM-CSF and 3H-TdR (0.5 microCi/ml). The following results were obtained as expressed by median values and ranges: 3H-TdR incorporation: 1.07 pmol/10(5) cells (0.0-10.1), TK: 7.3 pmol/min/mg protein (1.3-56.0), DCK: 9.3 pmol/min/mg protein (0.77-47.1), poly alpha: 1.7 pmol/min/mg protein (0.00-28.9), Ara-CTP: 53.3 ng/10(7) cells (13.3-211.0), 3H-Ara-C uptake: 0.06 pmol/10(5) cells (0.0-0.57). 3H-Ara-C uptake was correlated with 3H-TdR incorporation (r = 0.74) and with the (S-phase dependent) activities of TK (r = 0.73) and poly alpha (r = 0.71, but not with DCK activity or intracellular Ara-CTP content. Blast cells of 37 from 55 analyzed patients were found to be sensitive to GM-CSF stimulation as defined by an increase in 3H-TdR incorporation > or = 1.5-fold over control values after the 48 h GM-CSF exposure. In vitro data were related with clinical response to TAD-9 induction therapy in 43 patients with newly diagnosed AML, taking the blast cell reduction at day 10 or 16 to < 5% or > or = 5% residual blasts as early parameter for adequate or inadequate response, respectively. While neither 3H-Ara-C uptake, nor intracellular Ara-CTP concentration, TK nor DCK activity were predictive for response, a high 3H-TdR incorporation and a high poly alpha activity were associated with adequate blast cell reduction. Median values of 3H-TdR incorporation were 2.26 pmol/10(5) cells for patients with adequate blast cell clearance and 0.80 pmol/10(5) cells for patients with inadequate blast cell clearance (P = 0.11), the respective values for poly alpha were 3.22 pmol/min/mg protein for responders and 1.1 pmol/min/mg protein for non-responders (P = 0.0085).(ABSTRACT TRUNCATED AT 400 WORDS)

It has been reported that the adenine nucleoside analogue fludarabine is able to increase the phosphorylation and the cytotoxicity of cytosine arabinoside (Ara-C) in different leukemic models, both in vitro and in vivo. In poor prognosis acute myeloid leukemia (AML), the combination of fludarabine with Ara-C and granulocyte colony-stimulating factor (G-CSF) has proven to be a highly effective regimen. In this study we aimed to further investigate the effects of this drug combination. In vitro, on fresh AML cells from ten patients, our results confirm an additive cytotoxic effect displayed by fludarabine + Ara-C, as demonstrated by isobologram analysis of the data. The addition of G-CSF significantly increased the efficacy of the drug combination. These effects appeared to be related to an increased incorporation of [3H]Ara-C into cellular DNA in the presence of fludarabine + G-CSF. Furthermore, the quantitative evaluation of programmed cell death (apoptosis) showed that fludarabine + Ara-C + G-CSF induce apoptosis to a higher degree than either compound alone. This finding suggests that cooperative induction of apoptosis could be the potential mechanism of action of this drug combination.

Stimulation of clonogenic acute myeloid leukemia (AML) cells by hematopoietic growth factors (HGF) is capable of enhancing cytosine-arabinoside (Ara-C) cytotoxicity in vitro. Until now it has not been known to what extent in vitro Ara-C cytotoxicity can be restored by HGF stimulation in samples from previously treated AML patients. Therefore, we studied the individual effects of the hematopoietic growth factors (HGF) granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation on the Ara-C sensitivity of clonogenic leukemic cells from six patients with newly diagnosed AML. These results were directly compared with the outcome using AML samples from the same patients at relapse or primary refractory AML. In one patient, a sample of the second relapse was also studied. The results were expressed as IC50 values, and were used to calculate sensitivity ratios, defined as the ratio of the IC50 value with drug exposure alone and with HGF plus Ara-C. In AML samples treated with Ara-C alone and no HGF, IC50 values of Ara-C in relapsed/refractory AML were greater than IC50 values of AML cells at diagnosis. Addition of either HGF enhanced the Ara-C cytotoxicity for the relapsed samples significantly (for G-CSF p = 0.036, IL-3 p = 0.036, and for GM-CSF p = 0.036). The values of Ara-C sensitization of AML samples due to HGF at relapse did not significantly differ from those at diagnosis. However, enhancement of Ara-C cytotoxicity to AML progenitors by IL-3 or GM-CSF stimulation was significantly less in the cell specimens from AML recurrence patients as compared with the original diagnosis samples. In three AML samples at diagnosis and at their relapse, Ara-C incorporation into the DNA was determined. IL-3 stimulation enhanced Ara-C incorporation in all samples tested. Nevertheless, Ara-C incorporation in the relapsed samples was significantly less than that in the diagnosis samples of the same patients. A good correlation between Ara-C incorporation and bromodeoxyuridine (BrdU) incorporation was found. The results indicate that HGF stimulation in relapsed/refractory AML enhances Ara-C cytotoxicity, but not to the level that was observed with AML samples at diagnosis.

Patients with acute nonlymphocytic leukemia (ANLL) were treated by continuous infusion of ara-C (100 mg/m2/d x 10 days). During ara-C treatment, cellular arabinofuranosyl cytosine triphosphate (ara-CTP) pharmacokinetics was assessed in the circulating blasts of these patients using a high-performance liquid chromatography (HPLC) and an associated radioimmunoassay. Since a strong correlation was found between achievement of complete remission and cellular ara-CTP levels, we propose a calculation scheme that allows steady-state adjustment of ara-CTP levels during administration of ara-C. To improve the complete remission rate in patients with low ara-CTP levels, we sought optimum ara-C dosing. In order to achieve an optimal therapeutic response, in vivo ara-CTP formation has to be > 50 microM in leukemic cells. Conversely, using the same pharmacokinetic approach, the infusion rate at which to administer ara-C in order to reach in vivo ara-CTP concentration threshold and to achieve complete remission could be calculated for each patient.

The current study investigated the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the intracellular metabolism and cytotoxicity of 1-beta-D-arabinofuranosylcytosine (araC) in leukemic cells of 45 patients with acute myeloid leukemia (AML). AML blasts from bone marrow (BM) (n = 39) and peripheral blood (PB) (n = 17) were incubated for 48 h with or without GM-CSF (100 U/ml) followed by a concurrent treatment with increasing concentrations of araC (0.06-100 microM) for an additional 24 h. After GM-CSF a 1.5-8.4-fold (median 2.3) increase in 3H-araC incorporation into the DNA was observed in ten of 14 peripheral blast specimens and in 23 of 28 bone marrow samples, 18 of whom also showed an enhanced 3H-TdR incorporation (1.5-8.5-fold, median 2.0-fold). Four different types of response were identified when analyzing 3H-araC incorporation into the DNA of bone marrow samples in relation to the applied araC dose: (i) 8/28 cases had increases of the araC incorporation at all araC dose levels applied (0.06-100 microM), (ii) 12/28 at low araC concentrations only (0.06-1.0 microM), (iii) 3/28 at high araC concentrations only (10-100 microM), and (iv) 5/28 showed no increase at any dose level given. Hence, 20 of the 23 responding patients revealed a GM-CSF induced enhancement of araC incorporation at low or conventional doses of araC (0.06-1.0 microM). Fourteen of the 18 cases with concomitant rises of 3H-TdR and 3H-araC incorporation into the DNA after GM-CSF had elevated DNA polymerase alpha activity (16-531%, median 72%) and in ten cases overall DNA polymerase activity was enhanced (10-70%, median 22.5%). In contrast, thymidine kinase (TK) and deoxycytidine kinase (dCK) activity were elevated after GM-CSF in only ten and five patients, respectively. An increase in the fraction of cells in S phase was found in 11/21 bone marrow specimens and in 5/9 peripheral blast samples. However, no correlation was observed between increases in the proportion of cells in S phase and enhancements in enzyme activities. In 13 cases the cytotoxicity of araC with and without GM-CSF was assessed by means of a blast cell colony assay. Preincubation with GM-CSF increased the araC mediated cytotoxicity in ten of 13 patients by a median of 3.2-fold (range 2.2-229-fold). The respective LD50 values for araC were reduced from 0.45 to 0.19 microM on average.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of human recombinant GM-CSF (rGM-CSF) on the metabolism of high dose ara-C was determined in bone marrow mononuclear cells (BMMCs) from eight normal volunteers and from seven patients with acute myelogenous leukemia (AML). We incubated the cells with rGM-CSF alone, ara-C alone, or a combination of the two drugs. Treatment with rGM-CSF for 16 h increased the percentage of cells in S-phase both in normal BMMCs and leukemic marrow cells. The treatment with rGM-CSF alone produced an approximately two-fold increase in the intracellular dCTP pools in normal BMMCs, but this increment was not observed in leukemic marrow cells. Simultaneous exposure to rGM-CSF in combination with ara-C increased cytosine arabinoside triphosphate (ara-CTP) pools in leukemic blasts. In contrast, this treatment decreased ara-CTP pools in normal BMMCs. Moreover, when the cells were preincubated with rGM-CSF for 16 h prior to the exposure to ara-C, leukemic blasts achieved a 7-fold higher ara-CTP/dCTP ratio as compared with normal marrow cells. Treatment of the cells with rGM-CSF, either simultaneously or sequentially, resulted in significantly greater amounts of ara-C incorporation into DNA in leukemic marrow cells than normal counterparts. The higher accumulation of ara-CTP and subsequent increased incorporation of ara-C into DNA in leukemic cells treated with rGM-CSF lead to the enhanced ara-C-mediated inhibition of DNA synthesis as compared with normal BMMCs. The selective accumulation of ara-CTP in leukemic vs normal cells have implications for the efficacy of the treatment of AML patients with high dose ara-C and rGM-CSF.

Enhancement of the cytotoxicity of cytosine arabinoside (ara-C) by granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), and the mechanisms involved, were studied in the AML-193 human leukemia cell line. AML-193 cells require GM-CSF and G-CSF(CSFs) for optimum growth, and 24 h deprivation of CSFs decreased DNA synthesis measured in terms of 3H-thymidine incorporation. The DNA synthesis gradually recovered upon addition of CSFs. To examine the sensitivity to ara-C under different growth conditions, two groups of cell suspensions, one pretreated with CSFs after 24 h deprivation (CSFs(+) cells), and the other held continuously under CSFs-free conditions (CSFs(-) cells), were exposed to 1.0 microgram/ml of ara-C for 16 h. In clonogenic assays, CSFs(+) cells showed higher sensitivity to ara-C than CSFs(-) cells. These cell groups showed no significant difference in ara-C triphosphate accumulation or retention, though the amount of ara-C incorporated into the acid-insoluble fraction was two times greater in CSFs(+) cells than CSFs(-) cells, and that difference became even clearer in the retention pools. These data suggest that the enhancement of cytotoxicity by CSFs was due to the promotion of ara-C incorporation into DNA as a result of an increase of the cell fraction in the S phase.

The effects of the protein kinase C activator bryostatin 1, either with or without recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF) were examined with respect to the in vitro metabolism of ara-C in leukaemic myeloblasts obtained from 10 patients with acute myelogenous leukaemia (AML). Coincubation of cells with 12.5 x 10(-9) M bryostatin 1 and 10(-5) M ara-C for 4 h resulted in a significant increase in ara-CTP formation (compared to controls) in 6/10 specimens (mean increase 106%; range 38-255%), and no change in the remainder. In contrast, coincubation of cells with 1.25 ng/ml rGM-CSF resulted in a significant increase in only one specimen, and decreases in two. Bryostatin 1 also significantly increased ara-C DNA incorporation in 6/9 evaluable samples, including two which did not display an increase in ara-CTP formation. Coincubation of cells with both bryostatin 1 and rGM-CSF did not lead to a further increase in ara-CTP formation or ara-C DNA incorporation compared to values obtained with either agent alone. Finally, exposure of blasts to bryostatin 1 for 24 h before ara-C led to an increase in ara-CTP formation in 3/8 additional specimens, and a decrease in one sample displaying evidence of bryostatin 1-induced macrophage differentiation. Incubation of cells with both rGM-CSF and bryostatin 1 for this period resulted in ara-CTP levels equivalent to those obtained with bryostatin 1 alone. These studies indicate that while bryostatin 1 exerts a heterogeneous effect on ara-C metabolism in leukaemic myeloblasts, it is capable of potentiating ara-C phosphorylation in a subset of patient samples, including some that do not exhibit an increase in response to rGM-CSF. They also raise the possibility that bryostatin 1-induced potentiation of ara-C metabolism in some leukaemic cells may contribute, at least in part, to the antileukaemic efficacy of this drug combination.

Inhibitors of DNA synthesis, such as 1-beta-D-arabinofuranosylcytosine, have been proven useful in the management of acute myelogenous leukemia. Despite significant improvement of response rates, long-term disease-free survival can be presently achieved only in a small percentage of patients. As S-phase--specific drugs, such as the inhibitors of DNA synthesis, only reach the cycling part of their target populations, dormant cells are left mainly unaffected. Their survival, however, is largely responsible for later disease relapse. This paper reports experiments that show that cell kinetic manipulations by hematopoietic growth factors may help to overcome the problem of cytokinetic and pharmacologic resistance. Hematopoietic growth factors synergize with inhibitors of DNA synthesis given at low doses to induce a more mature phenotype or to enhance leukemia cell kill when combined with high doses of DNA inhibitors. In addition, we present data to unravel several aspects of the molecular mechanism underlying this synergism.

Exponentially growing K562 cells incubated with 1-beta-D-arabinofuranosylcytosine (ara-C) accumulate ara-C triphosphate (ara-CTP) at a higher rate and to a greater concentration after pretreatment with 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) than do cells treated with ara-C alone. Potentiation of ara-C metabolism is due in part to an indirect effect of F-ara-A triphosphate (F-ara-ATP)-mediated reduction in deoxynucleotide pools and consequent activation of deoxycytidine kinase. Because the levels of deoxynucleotide pools and the activity of deoxycytidine kinase are cell cycle-specific, we investigated the effect of cell cycle phases on the accumulation of ara-CTP and the influence of F-ara-A pretreatment on such accumulation. Exponentially growing K562 cells were fractionated into G1, S, and G2+M phase-enriched subpopulations (each enriched by > 60%) by centrifugal elutriation. The rate of ara-CTP accumulation was 22, 25, and 14 microM/h and the rate of F-ara-ATP accumulation was 38, 47, and 33 microM/h in the G1, S, and G2+M subpopulations, respectively. The rate of elimination of arabinosyl triphosphates was similar among the different phases of the cell cycle. After pretreatment with F-ara-A, the rate of ara-CTP accumulation in the G1, S, and G2+M phase-enriched subpopulations was 43, 37, and 26 microM/h, indicating a 1.7-, 1.5-, and 1.9-fold increase, respectively. These results suggest that a combination of F-ara-A and ara-C may effectively potentiate ara-CTP accumulation in all phases of the cell cycle. This observation is consistent with the results of studies on the modulation of ara-C metabolism by F-ara-A in lymphocytes and leukemia blasts obtained from patients with chronic lymphocytic leukemia and acute myelogenous leukemia, respectively.

The blast cells of acute myeloblastic leukemia (AML) usually require growth factors for optimum proliferation in cell culture. Growth factors also affect the sensitivity of AML blast cells to cytosine arabinoside (ara-C). Others have reported that factor-treated cells are more ara-C sensitive than blasts in culture without factors. These authors have reported previously that AML blasts grown with rG-CSF, with or without GM-CSF, are more sensitive than cells in GM-CSF alone. This paper reports experiments which show that changes in the ara-C sensitivities of blast cells in different growth factors are not explained by changes in the percentage of cells in the DNA synthesis (S) phase of the cycle. Blasts freshly obtained from five AML patients were cultured in either rG-CSF, rGM-CSF, or rIL-3; they were then exposed to 20 min pulses of either high specific activity tritiated thymidine (3HTdR) or a high concentration of ara-C. Regardless of the factor present, the pulse of 3HTdR decreased the number of clonogenic cells by about 50%, the result expected for actively proliferating cells with an S phase occupying about half the cycle time. The same result was found for four of the five blast cell populations grown in G-CSF and pulsed with ara-C; in contrast, clonogenic cells grown in GM-CSF or IL-3 from these four populations were not killed by ara-C. The blasts from the fifth patient were ara-C resistant under all conditions. It was concluded that exposure to GM-CSF or IL-3 decreased ara-C sensitivity in blasts that were actively making DNA. The observation was explored in more detail using a cell line (OCI/AML-1a) that is both ara-C sensitive and growth factor dependent. These studies showed that about 15 h of growth in factor are required for a change in ara-C sensitivity.

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 blast cells in acute myeloblastic leukemia (AML) respond to many of the same regulatory mechanisms that control normal hemopoiesis. These include the growth factors that bind to membrane receptors and steroid hormones or vitamins that have intracellular receptors. We report the effects in culture of the steroid glucocorticoid hydrocortisone on freshly explanted AML blasts from patients and on two continuous AML cell lines. Only small changes in clonogenic cell numbers in suspension cultures were seen in the presence of hydrocortisone. The most striking effect of the hormone was on the sensitivity of blasts cells to cytosine arabinoside (ara-C). In contrast to the response of AML blast cells to retinoic acid, a ligand for intracellular steroid receptors that sensitizes some blast populations to ara-C, hydrocortisone reduced the toxic effects of the drug. The protective action of hydrocortisone was not mediated through the cell cycle since exposure of blasts to hydrocortisone did not affect the percentage of cells in DNA synthesis as measured with the tritiated thymidine (3HTdR) "suicide" technique. The hydrocortisone effect could be demonstrated using a pulse (20 min) exposure protocol. Blasts pulsed with increasing specific activities of 3HTdR showed the usual response pattern with an initial loss in plating efficiency to about 50% of control, followed by a plateau, regardless of whether the cells had been exposed to hydrocortisone. Control blasts exposed to increasing ara-C concentrations gave very similar dose-response curves; in striking contrast, blast cells cultured in hydrocortisone, then pulsed with ara-C did not lose colony-forming ability even though the same population was sensitive to 3HTdR. The hydrocortisone effect was dose and time related; protection from ara-C increased from 10(-8) to 10(-5) M and was seen after 4 hr exposure but required 8 hr to reach a maximum. We conclude that hydrocortisone can protect blasts from the lethal effects of ara-C even while the cells are in active DNA synthesis.

Though data from cell lines are abundant, the reason for the development of resistance to 1-beta-D arabinofuranosylcytosine (ara-C) in vivo remains unresolved. A broad interpatient variation of metabolic parameters has further complicated interpretation of the results. The present study compares ara-C metabolism in leukemic blasts of two patients with newly diagnosed disease, before and after repeated treatment with ara-C containing chemotherapy regimens in vivo. Membrane transport of ara-C was unchanged after treatment. In addition, cell-free extracts of blasts obtained after treatment failure showed an unchanged cytidine deaminase activity. Though deoxycytidine kinase activity in cell extracts was unaltered or increased after treatment failure, the activity in situ, measured as the rate of 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) formation, was decreased. This could be shown to be due to an expansion of the deoxycytidine triphosphate (dCTP) pool. The severalfold increase in dCTP pool was accompanied by a decrease in thymidine triphosphate (dTTP) pool and correlated with a decrease in deoxycytidylate deaminase (dCMP-deaminase) activity in cell free extracts. Low dCMP-deaminase activity had been shown to confer an ara-C resistant phenotype to cell lines in vitro. Data presented in this paper show that a selection for leukemic blasts with low dCMP-deaminase activity can also be favored by ara-C containing treatment regimens in vivo. Our data suggest that this mechanism might contribute to treatment failure.

Human acute myelocytic leukemia (AML) marrow cells respond to stimulation with increased proliferation and enhanced intracellular metabolism of the cytotoxic antimetabolite 1-B-D arabinofuranosylcytosine (ara-C). Our previous studies have focused on the drug-induced humoral stimulatory activity (HSA) present in serum following initial cytoreduction which augments in vitro growth and biochemical pharmacology. The activity of HSA likely relates to the presence of multiple stimulators. The effect of 18-hr culture in purified recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) (1 ng/ml) on in vitro AML marrow cell [3H]dThd incorporation into DNA, intracellular ara-C activation to the triphosphate form (ara-CTP), and subsequent ara-CTP retention were determined in leukemic cells of 11 patients and compared with cells similarly cultured in HSA-containing sera. The stimulatory effects of rhGM-CSF and HSA on both growth and pharmacologic parameters were comparable for each AML population, with maximal response to both regulators detected for FAB M2. These data demonstrate that GM-CSF acts similarly to HSA as an active stimulator of leukemic cell proliferation and net intracellular ara-C metabolism in vitro, and support clinical trials designed to examine the role of rhGM-CSF in enhancing ara-C cytotoxicity by increasing the growth fraction of drug-responsive target cells in vivo.

Bone marrow cells of five patients with acute myeloid leukemia were fractionated by means of counterflow centrifugation (elutriation). The different fractions were enriched with cells belonging to subsequent stages of the cell cycle. Cytokinetic evaluation of these cell fractions was performed by [3H]thymidine autoradiography, [3H]thymidine incorporation and DNA/RNA-flow cytometry. Phosphorylation of cytosine arabinoside (ara-C, 1-beta-D-arabinofuranosylcytosine) in the different fractions was measured by incubation of the cells for 30 min with 1.07 microM [3H]ara-C. Phosphorylation of ara-C in the whole bone marrow samples ranged from 5.9 to 33.2 pmol/10(6) cells. In the fractions containing only G1-phase cells, phosphorylation ranged from 1.2 to 19.5 pmol/10(6) cells. The phosphorylation seems to increase before DNA synthesis starts. Maximal activities were found in the fractions enriched with cells in late G1- or S-phase of the cell cycle. In these fractions the ara-C phosphorylating activity was 1.5-8 times higher compared to the fractions with the lowest activity. One may therefore assume that not only S-phase cells are killed by ara-C, but that G1-phase cells which can phosphorylate ara-C, may also be doomed when they enter S-phase, since the elimination of the intracellular cytosine arabinoside tri-phosphate (ara-CTP) is a relatively slow process. The fraction of G1-phase cells phosphorylating ara-C, may be an important determinant in the extent of the cell-killing effect of ara-C treatment in the different leukemias.

1 The pharmacokinetics of subcutaneous cytosine arabinoside were compared with bolus intravenous injection and intravenous infusion in five patients with acute myelogenous leukaemia. 2 Subcutaneous cytosine arabinoside was rapidly absorbed and then declined biexponentially with initial and terminal half-lives similar to intravenous bolus injection. 3 Cytosine arabinoside levels declined rapidly after intravenous bolus and subcutaneous bolus injection, and fell below steady state infusion levels after a mean time of 40 min (intravenous bolus) and 100 min (subcutaneous injection).

1 The pharmacokinetics of cytosine arabinoside were studied after a single i.v. bolus of 2 mg/kg ara-C in patients with newly diagnosed untreated AML, using a bioassay and GC-MS method to measure the plasma concentrations. 2 Most patients showed a bi- or tri-phasic decline in plasma concentrations with time. Plasma clearance was 3.9 to 18.1 l/min as measured by the GC-MS method, and terminal half-lives varied from 7--107 min. 3 There was poor correlation of the GC-MS assay with the bioassay, probably because the latter was interfered with by the release of endogenous nucleosides from blasts after after ara-C. 4 Plasma concentrations were measured by GC-MS during continuous infusions in 14 patients. Plasma clearances were much lower than after a bolus, 0.39 to 5.25 l/min. 5 There was no correlation of response (remission or fall in peripheral blast count) with exposure to ara-C calculated from infusion dose, clearance and duration of infusion. 6 This study shows that ara-C pharmacokinetics varies markedly from patient to patient and that there is a wide range in the plasma concentrations associated with therapeutic response.

Arabinosylcytosine (ara-C) was administered by prolonged intravenous infusion with a portable liquid infusion system (LIS) to patients with acute myelogenous leukemia. With the use of tritiated ara-C and this portable system, pharmacologic studies were performed in 8 patients. Most of the plasma radioactivity is in the deaminated product, arabinosyluracil (ara-U). After continuous intravenous infusion, a constant ara-C level is achieved slowly in the plasma. Unless a loading (priming) dose is administered immediately before beginning the infusion, a steady-state ara-C level cannot be achieved until 8 to 24 hr after the infusion. The infusion system has two mechanisms--one for giving a loading dose (3 ml/min) and the other for regular infusion at a rate of 0.5 to 2.0 ml/hr. If a loading dose is given before continuous infusion, a steady-state are-C level is achieved within an hour. The plasma ara-C disappearance curves are biphasic with a terminal half-life of 104 min, which is the same as that of a single injection. The cumulative urinary excretion after approximately 23 hr of infusion varied from 14% to 35% in different patients; more than 90% is ara-U and the remainder is ara-C. Our results have demonstrated that LIS can be used conveniently to sustain a constant plasma level of ara-C. The LIS increases mobility of both inpatients and outpatients and is particularly convenient for ambulatory patients.

The relationship between in vitro leukemic cell growth kinetics, intracellular biochemical pharmacology of 1-beta-D-arabinofuranosylcytosine (ara-C), and clinical response to ara-C-containing timed sequential therapy was examined in leukemic marrow populations from 62 adults with acute myelogenous leukemia (AML). Leukemic blasts from 45 previously untreated and 17 relapse patients were obtained before therapy, and cultured in autologous pretreatment serum (APS) and in serum containing drug-induced humoral stimulatory activity (HSA). While all cell populations cultured in HSA demonstrated increased proliferation, only growth-stimulated cohorts from those patients achieving complete remission with ara-C-based therapy demonstrated enhanced intracellular drug activation and active drug retention relative to cells maintained in APS, whereas cells from nonresponsive patients demonstrated no such HSA-induced increases in intracellular ara-C metabolism. In this in vitro model system, sensitive AML populations behave similarly to normal hematopoietic cohorts, with direct linkage of HSA-perturbed growth and net pharmacologic parameters, while refractory cohorts evince uncoupling of these determinants in the growth-stimulated state. This model identifies behavior patterns that may discriminate clinical sensitivity from clinical resistance and may serve to predict clinical outcome to timed sequential therapy with ara-C.