Sensitive Leukemia Cell Lines Research Articles

RNA:m5c/Methyltransferases Mediate Chromatin Organization and 5-Azacytidine Response/Resistance in Leukemia

Background: The roles of RNA modifications and RNA-modifying enzymes (writers and erasers) in the regulation of chromatin structure and drug response/resistance in leukemia cells are not well understood. Most published studies have been focused on RNA N6-adenosine methylation (RNA:m6A) and its writers and erasers (Li, S., and Mason, C.E., 2014, Annu Rev Genomics Hum Genet 15, 127). Comparatively little is known about the functions of RNA cytosine methylation (RNA:m5C) and its writers and erasers (Gilbert, et al., 2016, Science 352, 1408). To date, ten RNA:m5C methyltransferases (RCMTs) have been identified, including NSUN1 to NSUN7, three versions (A, B and C) of NSUN5 and DNMT2, but the functions of RNA:m5C and RCMTs remains largely unknown (Schapira, M. 2016. ACS Chem Biol 11, 575). This study aims to determine the impact of RNA:m5C/RCMTs on chromatin structure and 5-azacytidine (5-AZA) response/resistance in myeloid leukemia cells.Experiments and Results: Our initial experiments demonstrate a clear relationship between drug resistance and the backbone chemical structures of nucleic acid analogues/drugs and distinct cell lineage-specific drug response patterns in our leukemia cell lines. These results led us to study the roles of RNA:m5C and RCMTs in the regulation of cell lineage-specific chromatin structure and differential drug response in leukemia. Our data demonstrate a marked increase in RNA:m5C and RCMTs as well as their associated active chromatin in our 5-AZA resistant leukemia cell lines, compared to the original 5-AZA sensitive leukemia cell lines. Our RNase-coupled co-immunoprecipitation demonstrates that RCMTs interact with different partners to form functional complexes and distinct chromatin structures, which are essential for the differential drug response and the survival of the leukemia cells. Specifically, NSUN3 and DNMT2 directly interact with a conserved RNA-binding protein hnRNPK to form a 5-AZA-sensitive multi-protein complex. hnRNPK preferentially binds methylated RNA and interacts with multiple proteins, including cell lineage-specific transcription factors GATA1 and SPI1/PU.1 and CDK9/P-TEFb, a kinase phosphorylating the serine 2 (S2) in the C-terminal domain (CTD) of RNA polymerase II (RNA-pol-II). In contrast, NSUN1 and NSUN2 are not associated with hnRNPK directly. Instead, NSUN1 directly binds active (phosphorylated) RNA-pol-II to form active chromatin structure that is completely resistant to 5-AZA. Strikingly, 5-AZA-resistant leukemia cells have a massive increase in NSUN1-and BRD4-associated active RNA-pol-II, compared to the original 5-AZA-sensitive leukemia cells. By employing the newly developed technologies, including 5-ethynyl uridine-clicking chemistry and ligation-mediated rolling circle amplification, we demonstrate that RCMTs recruit active RNA-pol-II to nascent (methylated) RNA to form distinct chromatin structures in the 5-AZA-sensitive vs. 5-AZA-resistant leukemia cells. Knockdown of the expression of these RCMTs causes growth inhibition in both the 5-AZA-sensitive and 5-AZA-resistant leukemia cells and also resensitize the 5-AZA-resistant leukemia cells to a low dose of 5-AZA. Based on our data, we propose the working model of RNA:m5C/RCMT-mediated chromatin structures in 5-AZA-sensitive vs. the 5-AZA-resistant leukemia cells (Figure 1). To test the working model, we applied various CDK and BRD4 inhibitors that target the serine phosphorylation in the CTD of RNA-pol-II to our 5-AZA-sensitive and 5-AZA-resistant-resistant leukemia cell lines. Our data show that these inhibitors synergize with 5-AZA and resensitize the 5-AZA-resistant leukemia cells, leading to growth inhibition in the leukemia cells at very low drug concentrations. By applying these technologies to clinical specimens, we demonstrate the existence of RNA:m5C/RCMT-mediated chromatin structures in clinical MDS/AML cells, supporting their importance and clinical relevance of our working model (Figure 1).Conclusion: Our data demonstrate that RCMTs interact with RNA-pol-II complex to form distinct chromatin structures at nascent (methylated) RNA, which regulate drug response/resistance in leukemia cells. Such RNA:m5C/RCMT-mediated drug-responsive chromatin structures may lead to the identification of new therapeutic avenues. [Display omitted] DisclosuresShammo:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Larson:Astellas: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen Inc.: Research Funding.

1,2,3-Triazole-Chalcone hybrids: Synthesis, in vitro cytotoxic activity and mechanistic investigation of apoptosis induction in multiple myeloma RPMI-8226

A new series of 1,2,3-triazole-chalcone hybrids has been synthesized and screened in vitro against a panel of 60 human cancer cell lines according to NCI (USA) protocol. Compound 4d having 3, 4-dimethoxyphenyl chalcone moiety, the most potent derivative, inhibited the growth of RPMI-8226 and SR leukemia cell lines by 99.73% and 94.95% at 10 μM, respectively. Also, it inhibited the growth of M14 melanoma, K-562 leukemia, and MCF7 breast cancer cell lines by more than 80% at the same test concentration. 4d showed IC50 values less than 1 μM on six types of tumor cells and high selectivity index reached to 104 fold on MCF7. Compound 4d showed superior activity than methotrexate and gefitinib against the most sensitive leukemia cell lines in addition to higher or comparable activity against the rest sensitive cell lines. Flow cytometry analysis in RPMI-8226 cells revealed that compound 4d caused cell cycle arrest at G2/M phase and induced apoptosis in a dose dependant manner. Mechanistic evaluation referred this apoptosis induction to triggering mitochondrial apoptotic pathway through inducing ROS accumulation, increasing Bax/Bcl-2 ratio and activation of caspases 3, 7 and 9.

NK-18, a promising antimicrobial peptide: anti-multidrug resistant leukemia cells and LPS neutralizing properties

With the increase of multidrug resistance, novel anti-leukemia agents with diverse mechanisms of action are required to address this challenge. NK-18, the core region of mammalian derived protein NK-lysin, effectively inhibited the viability of both multidrug resistant and sensitive leukemia cell lines. Meanwhile, this proliferation inhibition effect was not distinct between sensitive and multidrug resistant leukemia cell line. NK-18 showed selectivity between non-tumorigenic and tumorigenic cells. It preferentially bound to tumor cells whose outer leaflet with high phosphatidylserine content. NK-18 acted on the multidrug resistant leukemia cell line by a rapid pore formation on the cell membrane, it is not easy for K562/ADM cells developing resistance against NK-18. Furthermore, NK-18 could neutralize lipopolysaccharides by electrostatic attraction and reduce NO production. These research data demonstrated NK-18 possesses great advantage in the multidrug resistant leukemia treatment compared with conventional chemotherapies and it could be a potential candidate for further research.

Anti-Leukemia Effect of FF-10501-01, a Novel Inosine 5'-Monophosphate Dehydrogenase Inhibitor, in Advanced Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS), Including Hypomethylating Agent (HMA) Failures

Inosine 5'- monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes de novo synthesis of the guanine nucleotide and is overexpressed in both hematologic and solid tumors. FF-10501-01 is a potent new competitive IMPDH inhibitor. We investigated the anti-leukemia effect of FF-10501-01 in AML cell lines and in a Phase 1 clinical study in advanced AML and MDS, including HMA failures.Thirteen leukemia cell lines were studied, including 5 parental AML cell lines and their HMA-resistant derivatives (MOLM13, SKM1, HL60, TF1, and U937), and 3 other AML cell lines (KG1, HEL, and OCI-AML3). Cell proliferation was determined using trypan blue analysis. Flow cytometry was performed to detect drug-induced apoptosis and cell cycle analysis. High-performance liquid chromatography (HPLC) was performed to detect the intracellular concentrations of guanine nucleotides. Mycophenolic acid-treated cells were used as positive control. Effect of guanosine supplement on FF-10501-01 treatment was evaluated. Within 72 hours of treatment, FF-10501-01 inhibited proliferation of all 13 AML cell lines. The IC50 of FF-10501-01 ranged between 4.3 and 144.5 µM. MOLM13 was the most sensitive leukemia cell line, whereas the decitabine-resistant TF1 cell line was the most resistant. FF-10501-01-induced apoptosis was observed in all cell lines. Increased numbers of cells in G1 phase and decreased numbers in S phase were observed in MOLM13, SKM1 and TF1 cell lines treated with <100 µM FF-10501-01. Decreased intracellular concentrations of guanine nucleotides were observed in MOLM13 and SKM1 cell lines treated with 3 to 30 µM of FF-10501-01 for 24 hours. Proliferation was partially rescued after 72 hours of treatment with 3 µM guanosine and FF-10501-01 in MOLM-13, HL60 cells and their HMA-resistant derivatives. No treatment synergy was observed with the combination of FF-10501-01 with HMAs in MOLM-14 and HL-60 or their HMA-resistant cell lines.In summary, FF-10501-01 produced potent anti-proliferative and apoptotic effects on AML cell lines through inhibition of de novo guanine nucleotide synthesis.In view of these pre-clinical findings, we performed a standard 3+3 dose-escalation Phase 1 trial to access the safety and clinical activity of FF-10501-01 in patients with advanced AML, MDS and chronic myelomonocytic leukemia (CMML). Eligibility criteria: age ³ 18 years, high risk MDS/CMML, AML with documented PD following previous therapy, AML ≥ 60 years of age and not a candidate for other therapy, adequate renal and hepatic function, and no known history of significant cardiac disease. Sixteen patients (12 AML, 4 MDS) have been enrolled in 5 dose cohorts (50 - 400 mg/m2 PO BID) for 14 days on/14 days off each 28-day cycle, including 8 M and 8 F. Median (range) values: age 75.3 yrs (59.1 - 88.6); bone marrow blasts for AML patients 40.5% (12 - 71), for MDS patients 10% (6 - 13), or 30% overall (6 - 71); and prior treatment regimens 2.5 (1 - 6). All patients relapsed from, or progressed on, prior HMAs. Mutations in FLT3, NPM1, GATA2, TET2, ASXL1, DNMT3A and/or MDM2 were present in 4/16 (25%) patients.The median number of FF-10501-01 cycles received to date is 1.5 (range 1 - 10). No DLTs or drug-related serious adverse events (AEs) have been observed and FF-10501-01 has been very well tolerated through 5 - 10 cycles. The most frequent drug-related AEs have been Gr 1-2 nausea, diarrhea and fatigue. Drug-related Gr 4 prolonged thrombocytopenia and Gr 4 prolonged neutropenia were reported in one patient at 200 mg/m2 BID.Two partial responses (PRs) have been achieved in 1 patient each at 50 and 100 mg/m2 BID after 3 cycles, 7 (50%) patients demonstrated long-term stable disease over 2 - 10 cycles, and 4 patients have remained on study drug through 5 - 10 cycles and are still ongoing. Updated safety and efficacy data, including PK/PD, will be presented at the meeting.FF-10501-01 is a promising new agent for the treatment of advanced AML and MDS. Preclinical activity was seen in multiple leukemia cell lines. In a Phase 1 trial, clinical activity with PRs, prolonged disease stabilization and a highly tolerable safety profile were observed. The Phase 2 expansion phase will be initiated soon. DisclosuresDiNardo:Novartis: Research Funding. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Smith:Westat Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Gipson:Strategia Therapeutics, Inc.: Employment. Rosner:Strategia Therapeutic, Inc.: Employment. Madden:Strategia Therapeutics, Inc.: Employment. Myers:Strategia Therapeutics, Inc.: Employment. Paradiso:Strategia Therapeutics, Inc.: Employment.

A-Type Proanthocyanidins Prevent Engraftment Of Primary Acute Myelogenous Leukemia Cells In Mice and Exhibit Potentially Novel Anti-Leukemia Mechanisms

Acute myelogenous leukemia (AML) is often a fatal disease where after strong induction therapy most patients relapse and die. AML originates and is maintained by leukemia stem cells (LSCs). Failure to eliminate LSCs by chemotherapy is likely to result in disease relapse. Therefore, it is a priority to identify new therapies that eliminate blasts while ablating LSCs and preventing a relapse.We have found that a unique class of compounds in cranberries (Vaccinium macrocarponAit.), known as A-type proanthocyanidins (A-PACs), were effective against several leukemia cell lines and primary AML samples in vitro. A-PACs consist of monomeric epicatechin units attached to one another by a carbon-carbon bond and a distinctive ether bond that differentiates these compounds from other proanthocyanidins found in nature. Moreover, A-PACs possess ortho-hydroxyl phenolic groups that have the potential to bind to iron and alter redox status.Preliminary work showed that pre-treatment with antioxidants or holo-transferrin (iron-saturated transferrin) partially protected AML cells from A-PAC induced cell death (p<0.01). A-PACs were also found to selectively ablate leukemia stem and progenitor cells, with minimal effects on normal hematopoetic stem cells. Furthermore, AML engraftment of cells treated ex vivo with 62.5 µg/ml A-PACs was decreased (90.6%, n=3, p<0.001), while normal CD34+ cells retained engraftment capability in immunodeficient mice. It was also found that a fraction of A-PACs of up to 7 degree of polymerization was more effective than individual A-PACs. This information prompted us to investigate the in vivo anti-leukemia effects of A-PACs in xenotransplanted mice with primary AML samples, and to further investigate the mechanisms associated with these compounds.Primary AML cells were injected in sub-lethally irradiated NOD/SCID mice. Four weeks after injections, when human leukemia cells have engrafted, intraperitoneal injections of cytarabine (AraC) at 60 mg/kg were given to the mice for 1 week everyday or A-PACs (100 mg/kg dose every 3 days for A-PACs) and vehicle control (1% DMSO in PBS every 3 days) were injected for 2.5 weeks. Mice were sacrificed and leukemia engraftment evaluated using anti-human CD45 and CD33. Moreover, primary cells treated with A-PACs were assessed for effects on iron metabolism, ROS, and survival pathways either by gene expression analysis, flow cytometry or mass spectrometry.Administration of A-PACs to NOD-SCID mice bearing AML tumors reduced tumor burden. Mice that were treated with the vehicle control had engraftment of AML primary cells equivalent to 16.1% (95% CI: -6.0, 38.37; n=4), whereas the mice treated with the A-PACs and AraC showed a level of engraftment of 4.9% (95% CI: 2, 8; n=5) and 5.8% (95% CI: -1.1, 12.7; n=5), respectively. No significant changes in hemoglobin or weight were found between the different treatment groups. Moreover, qPCR analysis of sensitive leukemia cell lines treated with A-PACs showed changes in gene expression of several iron metabolism genes in sensitive leukemia cell lines (up-regulation of ferritin and transferrin receptors 1 and down-regulation of ferroportin) and several ROS-relevant genes (down-regulation of nuclear factor erythroid-2-related factor 2 and glutamate-cysteine ligase regulatory subunit). Mass spectrometry also confirmed that A-PACs bind iron.The results indicate that A-PACs not only target primary AML cells in vitro but are also effective in vivo. Secondary transplants are also being performed to determine the effects on LSC activity. Some of the anti-leukemia mechanisms under investigation include effects related to iron metabolism, ROS or inhibition of survival pathways. Understanding the unique structure and biological effects of A-PACs may provide novel information about pathways involved in the survival of LSCs and provide crucial information in preparation for clinical trials and/or optimal combination drug therapies. Disclosures:Rivella:Novartis: Consultancy; Bayer: Consultancy; Isis: Consultancy, Research Funding; Merganser: Equity Ownership, Research Funding; Biomarin: Consultancy; Alexion: Consultancy; Imago: Consultancy.

Edelfosine lipid nanosystems overcome drug resistance in leukemic cell lines

Although current therapies have improved leukemia survival rates, adverse drug effects and relapse are frequent. Encapsulation of edelfosine (ET) in lipid nanoparticles (LNs) improves its oral bioavailability and decreases its toxicity. Here we evaluated the efficacy of ET-LN in myeloid leukemia cell lines. Drug-loaded LN were as effective as free ET in sensitive leukemia cell lines. Moreover, the encapsulated drug overcame the resistance of the K562 cell line to the drug. LN containing ET might be used as a promising drug delivery system in leukemia due to their capacity to overcome the in vivo pitfalls of the free drug and their efficacy in vitro in leukemia cell lines.

Leukemic cell xenograft in zebrafish embryo for investigating drug efficacy

Zebrafish were proposed as an alternative to mammalian models to assess the efficacy and toxicity of antileukemic drugs. Due to the limited number of transgenic zebrafish leukemia models, we explored human leukemic cell xenograft in zebrafish embryos. Human leukemic cell lines and blast cells sorted from patients with acute myelogenous leukemia were injected 48 hours post-fertilization and remained in the circulation of zebrafish embryos for several days without affecting their development. Imatinib and oxaphorines did not demonstrate any toxicity on normal zebrafish embryos and decreased the leukemic burden in animals xenografted with sensitive leukemic cell lines. Two other molecules, all-trans retinoic acid and the translation inhibitor 4EGI-1, demonstrated teratogenic effects at concentrations shown to be efficient in vitro, which precluded investigation of their antileukemic activity in such models. Altogether, xenografted leukemic cells in zebrafish embryos are a pharmacologically relevant model for screening non-teratogenic drugs.

Abstract A41: Somatic mutations in human cancer cell lines predict sorafenib response

Abstract It is now widely accepted that a spectrum of somatic alterations occur in human cancer and that particular alterations are associated with response or lack of response to anti-cancer therapies. Here, we undertook a systematic correlation analysis of somatic mutations, copy number aberrations and drug response across 300+ human cancer cell lines. We studied the RAF and VEGFR inhibitor, sorafenib, evaluating associations between sorafenib response and somatic mutations in 35 common cancer genes and high-level amplifications and homozygous deletions in more than 100 minimal common regions of gain and loss, which include many known oncogenes and tumor suppressor genes. Notably, we found that NRAS mutations, among other alterations, were significantly associated with sensitivity to sorafenib (odds ratio = 7, p-value = 0.003), whereas activating BRAF mutations were strongly associated with a lack of response (odds ratio = 4.9, p-value = 0.012). Specifically, 64% (7 of 11) of NRAS mutant cell lines were sensitive to sorafenib, while only 20% (34 of 170) of NRAS wild-type lines were sensitive. On the contrary, just 6.7% (2 of 30) of BRAF mutant lines were sensitive to sorafenib. Because NRAS mutations occur frequently in leukemia and no leukemia cell lines were included in the original analysis, we selected 5 NRAS mutant and 5 NRAS wild-type leukemia cell lines for further evaluation. Consistent with our previous observation, the four most sensitive leukemia cell lines were all NRAS mutant, each line exhibiting greater than 50% growth inhibition after 72 hours of exposure to 4μM sorafenib. These results confirm the original association and suggest that across both solid tumors and hematologic malignancies, NRAS mutations may identify a subset of cancers that are likely to respond sorafenib. In addition, this work demonstrates that systematic analysis of somatic alterations and drug response across a large collection of cancer cell lines is capable of discovering novel predictive biomarkers. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A41

Abstract A47: Somatic mutations in human cancer cell lines predict sorafenib response

Abstract It is now widely accepted that a spectrum of somatic alterations occur in human cancer and that particular alterations are associated with response or lack of response to anti-cancer therapies. Here, we undertook a systematic correlation analysis of somatic mutations, copy number aberrations and drug response across 300+ human cancer cell lines. We studied the RAF and VEGFR inhibitor, sorafenib, evaluating associations between sorafenib response and somatic mutations in 35 common cancer genes and high-level amplifications and homozygous deletions in more than 100 minimal common regions of gain and loss, which include many known oncogenes and tumor suppressor genes. Notably, we found that NRAS mutations, among other alterations, were significantly associated with sensitivity to sorafenib (odds ratio = 7, p-value = 0.003), whereas activating BRAF mutations were strongly associated with a lack of response (odds ratio = 4.9, p-value = 0.012). Specifically, 64% (7 of 11) of NRAS mutant cell lines were sensitive to sorafenib, while only 20% (34 of 170) of NRAS wild-type lines were sensitive. On the contrary, just 6.7% (2 of 30) of BRAF mutant lines were sensitive to sorafenib. Because NRAS mutations occur frequently in leukemia and no leukemia cell lines were included in the original analysis, we selected 5 NRAS mutant and 5 NRAS wild-type leukemia cell lines for further evaluation. Consistent with our previous observation, the four most sensitive leukemia cell lines were all NRAS mutant, each line exhibiting greater than 50% growth inhibition after 72 hours of exposure to 4uM sorafenib. These results confirm the original association and suggest that across both solid tumors and hematologic malignancies, NRAS mutations may identify a subset of cancers that are likely to respond sorafenib. In addition, this work demonstrates that systematic analysis of somatic alterations and drug response across a large collection of cancer cell lines is capable of discovering novel predictive biomarkers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A47.

Multiple Myeloma

A new series of 1,2,3-triazole-chalcone hybrids has been synthesized and screened in vitro against a panel of 60 human cancer cell lines according to NCI (USA) protocol. Compound 4d having 3, 4-dimethoxyphenyl chalcone moiety, the most potent derivative, inhibited the growth of RPMI-8226 and SR leukemia cell lines by 99.73% and 94.95% at 10 μM, respectively. Also, it inhibited the growth of M14 melanoma, K-562 leukemia, and MCF7 breast cancer cell lines by more than 80% at the same test concentration. 4d showed IC50 values less than 1 μM on six types of tumor cells and high selectivity index reached to 104 fold on MCF7. Compound 4d showed superior activity than methotrexate and gefitinib against the most sensitive leukemia cell lines in addition to higher or comparable activity against the rest sensitive cell lines. Flow cytometry analysis in RPMI-8226 cells revealed that compound 4d caused cell cycle arrest at G2/M phase and induced apoptosis in a dose dependant manner. Mechanistic evaluation referred this apoptosis induction to triggering mitochondrial apoptotic pathway through inducing ROS accumulation, increasing Bax/Bcl-2 ratio and activation of caspases 3, 7 and 9.

Modulation of the Glycolysis Pathway Reverses Prednisolone Resistance in Pediatric Acute Lymphoblastic Leukemia Cells

Although treatment of childhood acute lymphoblastic leukemia (ALL) has greatly improved over the past decades, resistance to the glucocorticoids prednisolone and dexamethasone still causes a serious drawback in the success rate of conventional chemotherapy. Resistance to prednisolone at initial diagnosis in particular is related to an unfavorable event free survival. In addition, in vitro prednisolone resistance is recognized as an important negative parameter for long-term clinical outcome, even in patients who initially have a good in vivo response to one week with glucocorticoids. Therefore, alternative therapies that reverse resistance towards prednisolone and dexamethasone are indispensable to increase treatment efficacy. However, the exact mechanisms that cause cellular resistance to glucocorticoids are currently unknown. A better understanding of the pathways that lead to glucocorticoid resistance will thus be essential to be able to improve treatment results. We previously identified new genes that could be associated with prednisolone resistance in pediatric ALL by gene expression profiling [Holleman et al., N Engl J Med 2004]. Many of these genes were linked to glucose metabolism and were found to be significantly upregulated in prednisolone-resistant precursor B-ALL cells (p≤0.001). Expression levels of hypoxia-inducible factor-1 alpha (HIF-1a), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), carbonic anhydrase 4 (CA4) and glucose transporter 3 (GLUT3/SLC2A3) were higher in prednisolone resistant cells when compared to prednisolone sensitive leukemia cells (fold change = 1.43, 1.26, 1.56 and 2.08 respectively). To investigate the causal relationship between the glycolysis pathway and prednisolone resistance, the glycolytic rate of various prednisolone-resistant and –sensitive leukemic cell lines was determined. The prednisolone-resistant cell lines Jurkat and Molt4 (LC50≥150) showed a higher glucose consumption compared to the prednisolone-sensitive cell lines Tom-1 and RS4;11 (LC50≤0.1), concordant with the microarray data. Several drugs that have been reported to affect glycolysis were subsequently tested for their ability to reverse resistance to glucocorticoids, using the in vitro MTT cytotoxicity assay. Treatment of the prednisolone-resistant cell lines Jurkat and Molt4 with 2-deoxyglucose (2-DG), Lonidamine or 3-Bromopyruvate increased the sensitivity to prednisolone and dexamethasone about ten-fold, while treatment of the prednisolone sensitive cell lines Tom-1 and RS4-11 had little or no effect. Importantly, the sensitizing effect of 2-DG on glucocorticoid resistance was not limited to cell lines, but was also observed in isolated primary leukemia cells being resistant to prednisolone whereas the effect was neglectable in sensitive cases. Moreover, downregulation of the expression of GAPDH by RNA interference also sensitized to prednisolone, comparable to treatment with glycolytic inhibitors. RNA interference for the transcription factor HIF-1a, that regulates the expression of genes involved in glycolysis, did not alter the sensitivity for prednisolone, indicating that the upregulation of HIF-1a is not causally related to glucocorticoid resistance in ALL. Together, these findings indicate the importance of the glycolysis pathway in glucocorticoid resistance in leukemia, and suggest that targeting this pathway is a viable strategy for modulating prednisolone resistance in ALL.

Growth inhibition, cytokinesis failure and apoptosis of multidrug-resistant leukemia cells after treatment with P-glycoprotein inhibitory agents.

The multidrug transporter P-glycoprotein (Pgp), which is frequently overexpressed in multidrug resistant leukemia, has many proposed physiological functions including involvement in transmembraneous transport of certain growth-regulating cytokines. Therefore, we studied cell growth of three pairs of drug resistant and sensitive leukemia cell lines (KG1a, K562 and HL60) exposed to three different inhibitors of Pgp. The resistant KG1a and K562 sublines, which expressed high levels of Pgp, responded to low doses of the cyclosporin SDZ PSC 833, the cyclopeptolide SDZ 280-446, and the cyclopropyldibenzosuberane LY335979 with a dose-dependent growth inhibition. In the resistant variants of KG1a and K562 cells the mean half-maximal growth inhibitory doses (GI50) of SDZ PSC 833 were 312 (SE 41) and 414 (SE 50) nM, those of SDZ 280-446 were 685 (SE 51) and 578 (SE 54) nM, and those of LY335979 were 66 (SE 1) and 48 (SE 8) nM, respectively. Exposure to 1 microM SDZ PSC 833 resulted in tetraploidization, cytokinesis failure and apoptosis of the KG1a and K562 MDR variants. Conversely, parental cells with no or low levels of Pgp and the non-Pgp resistant variant of HL60 cells were not receptive to these cytotoxic effects. We conclude that inhibition of Pgp may exercise selective cytotoxicity in Pgp-rich leukemia cells indicating a possible therapeutic target in multiresistant leukemia.

Characterization of Nuclear Retinoic Acid-Binding Activity in Sensitive Leukemic-Cell Lines - Cell-Specific Uptake of Atra and Rarα Protein Modulation

The diverse effects of all- trans retinoic acid (ATRA) on growth, differentiation and homeostasis of vertebrate organisms are mediated by three distinct isoforms of retinoic acid receptors (RARs), Although it is not known to what extent each RAR contributes to the different effects of ATRA, several studies have demonstrated that ATRA induced granulocytic differentiation in human myeloid leukemic cell lines is mediated by RARα. In this study, we investigated ATRA binding affinity of the endogenous nuclear receptors of HL-60 and NB4 leukemic cells, Scatchard plot analysis yielded an apparent dissociation constant of 5±0.3 nM and 1400±80 receptor sites per cell in HL-60 cells, whereas the NB4 promyelocytic leukemic cell line showed a lower affinity (8.5±0.5 nM and 900±30 receptor sites per cell). Modulation of RAR alpha protein (5 fold excess) was found in NB4 cells after 24 hours ATRA exposure, whereas HL-60 cells required a 72-hour culture period to weakly increase the RARα protein level These data were closely related to the ATRA intracellular concentration and kinetics of terminal differentiation of the cells.

Structure-dependent antitumor activities of novel anthracyclines YM1, YM3, YM4 and YM6: drug transport properties and effects on biomacromolecule synthesis in drug sensitive and resistant leukemia cells.

The antitumor activities of four novel doxorubicin (DOX) analogues, YM1, YM3, YM4 and YM6 in relation to their structure and drug transport properties, have been investigated in U937 monocytic and CCRF-CEM lymphoid drug sensitive leukemia cell lines, as well as in CEM/VLB100, a drug resistant subline displaying high levels of P-glycoprotein. Treatment of all cell lines with YM1, 3, 4 and 6 produced a dose-dependent decrease in DNA, RNA and protein synthesis as measured by [3H]-thymidine, [3H]-uridine and [3H]-leucine uptake respectively. YM1 was more effective than YM3, YM4 or YM6 against the drug sensitive cells. The antitumor effects of all these DOX-analogues on macromolecule synthesis in U937 and CCRF-CEM cells were lower than that of DOX and epirubicin (EDR). A rapid accumulation of the novel anthracyclines was found in all cell lines compared with DOX or EDR. However, the maximal accumulation of the DOX-analogues was lower than that of EDR. There is a greater efflux from CCRF-CEM sensitive cells and less from CEM/VLB100 resistant cells of the DOX-derivatives when compared with EDR and DOX. Drug-induced cytotoxicity significantly correlated (P < 0.05) with drug retention levels in CCRF-CEM and U937 drug sensitive cells as indicated by an inverse correlation curve between anthracycline retention and drug-induced IC50 value. It was demonstrated that an increased level of drug retained within the sensitive cells would therefore produce a more cytotoxic effect of the drug. However, no such correlation was observed in CEM/VLB100 resistant cells. YM3 was shown to have an increased antitumor activity against CEM/VLB100 resistant cells compared with DOX with a lower resistance factor. These results showed that the antitumor effects of four novel DOX-analogues, like DOX or EDR, were associated with inhibition of DNA replication, transcription and translation. The finding that resistant leukemic cells are more susceptible to the cytotoxic effect of YM3 than DOX warrants further investigation to identify the intrinsic mechanism of resistance.