Clinical Leukemia Samples Research Articles

SUCLG1 restricts POLRMT succinylation to enhance mitochondrial biogenesis and leukemia progression

Mitochondria are cellular powerhouses that generate energy through the electron transport chain (ETC). The mitochondrial genome (mtDNA) encodes essential ETC proteins in a compartmentalized manner, however, the mechanism underlying metabolic regulation of mtDNA function remains unknown. Here, we report that expression of tricarboxylic acid cycle enzyme succinate-CoA ligase SUCLG1 strongly correlates with ETC genes across various TCGA cancer transcriptomes. Mechanistically, SUCLG1 restricts succinyl-CoA levels to suppress the succinylation of mitochondrial RNA polymerase (POLRMT). Lysine 622 succinylation disrupts the interaction of POLRMT with mtDNA and mitochondrial transcription factors. SUCLG1-mediated POLRMT hyposuccinylation maintains mtDNA transcription, mitochondrial biogenesis, and leukemia cell proliferation. Specifically, leukemia-promoting FMS-like tyrosine kinase 3 (FLT3) mutations modulate nuclear transcription and upregulate SUCLG1 expression to reduce succinyl-CoA and POLRMT succinylation, resulting in enhanced mitobiogenesis. In line, genetic depletion of POLRMT or SUCLG1 significantly delays disease progression in mouse and humanized leukemia models. Importantly, succinyl-CoA level and POLRMT succinylation are downregulated in FLT3-mutated clinical leukemia samples, linking enhanced mitobiogenesis to cancer progression. Together, SUCLG1 connects succinyl-CoA with POLRMT succinylation to modulate mitochondrial function and cancer development.

Triptolide promotes ferroptosis by suppressing Nrf2 to overcome leukemia cell resistance to doxorubicin.

Doxorubicin (DOX) is an extensively used chemotherapeutic drug to treat leukemia. However, there remains a pivotal clinical problem of resistance to DOX in patients with leukemia. Erythroid 2‑related factor2 (Nrf2) is a master regulator of antioxidation response which serves a critical role in maintaining cellular oxidative homeostasis. However, whether Nrf2 is involved in DOX resistance is not totally clear. It is well‑documented that triptolide, a widely used drug to treat autoimmune disorders, possesses anti‑cancer activities, yet whether triptolide affects leukemia cell sensitivity to DOX remains to be elucidated. The present study aimed to determine the role of triptolide‑mediated downregulation of Nrf2 in regulating leukemia cell ferroptosis and resistance to DOX. For this purpose, low‑dose DOX was used to establish DOX‑resistant K562 cells and HL‑60 cells. Nrf2 mRNA and protein expression were examined by quantitative PCR and western blotting assays. The effects of triptolide on leukemia cell viability, reactive oxygen species (ROS) levels, or lipid oxidation were determined by CCK8 assay, DCFH‑DA assay, or BODIPY 581/591 C11 assay, respectively. The results show that Nrf2 expression was significantly upregulated in DOX‑resistant leukemia cells and clinical leukemia samples. Silencing of Nrf2 significantly sensitized leukemia cells to DOX. Furthermore, it was demonstrated that triptolide inhibited Nrf2 expression and induced leukemia cell ferroptosis, as evidenced by increased ROS levels and lipid oxidation as well as decreased glutathione peroxidase4 expression. Ectopic expression of Nrf2 significantly rescued triptolide‑induced leukemia cell ferroptosis. Notably, the present study showed that triptolide re‑sensitized DOX‑resistant leukemia cells to DOX. In conclusion, the present study indicated that Nrf2 served a critical role in leukemia cell resistance to DOX and triptolide‑induced ferroptosis and suggested a potential strategy of combination therapy using triptolide and DOX in leukemia treatment.

Drug ranking using machine learning systematically predicts the efficacy of anti-cancer drugs

Artificial intelligence and machine learning (ML) promise to transform cancer therapies by accurately predicting the most appropriate therapies to treat individual patients. Here, we present an approach, named Drug Ranking Using ML (DRUML), which uses omics data to produce ordered lists of >400 drugs based on their anti-proliferative efficacy in cancer cells. To reduce noise and increase predictive robustness, instead of individual features, DRUML uses internally normalized distance metrics of drug response as features for ML model generation. DRUML is trained using in-house proteomics and phosphoproteomics data derived from 48 cell lines, and it is verified with data comprised of 53 cellular models from 12 independent laboratories. We show that DRUML predicts drug responses in independent verification datasets with low error (mean squared error < 0.1 and mean Spearman’s rank 0.7). In addition, we demonstrate that DRUML predictions of cytarabine sensitivity in clinical leukemia samples are prognostic of patient survival (Log rank p < 0.005). Our results indicate that DRUML accurately ranks anti-cancer drugs by their efficacy across a wide range of pathologies.

β-catenin induces expression of prohibitin gene in acute leukemic cells

Prohibitin (PHB) is a multifunctional protein conserved in eukaryotic systems and shows various expression levels in tumor cells. However, regulation of PHB is not clearly understood. Here, we focused on the regulation of PHB expression by Wnt signaling, one of dominant regulatory signals in various leukemic cells. High mRNA levels of PHB were found in half of clinical leukemia samples. PHB expression was increased by inhibition of the MAPK pathway and decreased by activation of EGF signal. Although cell proliferating signals downregulated the transcription of PHB, treatment with lithium chloride, an analog of the Wnt signal, induced PHB level in various cell types. We identified the TCF-4/LEF-1 binding motif, CATCTG, in the promoter region of PHB by site-directed mutagenesis and ChIP assay. This β-catenin-mediated activation of PHB expression was independent of c-MYC activation, a product of Wnt signaling. These data indicate that PHB is a direct target of β-catenin and the increased level of PHB in leukemia can be regulated by Wnt signaling.

Metformin Sensitizes Leukemia Cells to Vincristine via Activation of AMP-activated Protein Kinase.

Vincristine is extensively used chemotherapeutic medicine to treat leukemia. However, it remains a critical clinical problem with regard to its toxicity and drug-resistance. AMP-activated protein kinase (AMPK) is an energy sensor that is pivotal in maintaining cell metabolic homeostasis. It is reported that AMPK is involved in vincristine-induced apoptosis. However, whether AMPK is involved in chemotherapy-resistance is largely unclear. It is well-documented that metformin, a widely used medicine to treat type II diabetes, possesses anti-cancer activities, yet whether metformin affects leukemia cell viability via vincristine is unknown. In this study, we showed that both AMPKα1 mRNA and phosphorylated AMPK protein levels were significantly decreased in clinical leukemia samples. We further demonstrated that metformin sensitized leukemia cells to vincristine-induced apoptosis in an AMPK-dependent manner. In addition, knockdown of AMPKα1 significantly reduced the effects of metformin on vincristine-induced apoptosis. Taken together, these results indicate that AMPK activation is critical in metformin effects on vincristine-induced apoptosis and suggest a putative strategy of a combination therapy using metformin and vincristine in treatment of leukemia.

Epigenetic regulation of putative tumor suppressor TGFBI in human leukemias

Background Both in vitro and in vivo data have demonstrated the TGFBI gene functions as a putative tumor suppressor and is frequently downregulated in human tumors of different histological types. The hypermethylation of the TGFBI promoter, as one of the main regulatory mechanisms, is associated with TGFBI silencing. In this study, we used a methylation-specific PCR (MSP) method to evaluate the methylation status of the TGFBI promoter in human leukemias. Methods Real-time RT-PCR and methylation-specific PCR approaches were performed to define the TGFBI expression and promoter methylation in human leukemia cell lines and clinical samples. Genomic DNA was isolated from peripheral blood mononuclear cells from leukemia patients, bisulfite-converted, and analyzed by the MSP method. Results Hypermethylation of the TGFBI promoter occurred in leukemia cell lines and demethylation treatment re-expressed TGFBI at a substantially increased level in most of leukemia cell lines tested. Furthermore, a much higher level of CpG island methylation and a significantly lower TGFBI expression were also identified in clinical leukemia samples. Conclusion The results suggest an important role of promoter methylation in regulating TGFBI expression in leukemia, which provides a useful diagnostic marker for clinical management of human leukemias.

Disease-Associated Chromatin Conformation and Therapeutic Implications In Leukemia

Disease- or treatment-associated chromatin conformation has yet to be illustrated. Here we first demonstrate that monocytic and erythroid leukemia cell lines have distinctly different chromatin conformation at the PU.1 locus: a looped, connected, RNA-polymerase-II-bound, active conformation in the former and a disconnected, inactive conformation in the latter. These conformations undergo opposite transformations, becoming more active in the erythroid leukemia line and less active in the monocytic leukemia line, in response to both DNA and histone hypomethylating drugs.To explore the underlying mechanisms we developed a novel method to analyze DNA modifications. We demonstrate that the erythroid leukemia line has a marked drug-responsive change in both hydroxymethyl-CpG and 5-meythyl-CpG at the promoter, whereas the monocytic leukemia line has a higher level bivalent histone/chromatin with co-localized H3K4me3 and H3K27me3 at the enhancer. Consequently, the erythroid leukemia line is more sensitive to DNA hypomethylation while the monocytic leukemia line is more sensitive to histone hypomethylation. Further studies on clinical leukemia samples confirm the findings, and demonstrate that the leukemic cells from the clinical samples have different chromatin conformations with much more bivalent chromatin and denser DNA/histone modifiers and are more sensitive to hypomethylating drugs, compared to normal controls. Different chromatin conformations dictate preferential drug responses and open new diagnostic and therapeutic avenues. Disclosures: No relevant conflicts of interest to declare.

Abstract 4122: Ras GTPase activating like protein (IQGAP1) in human acute myelogenous leukemia.

Abstract Background and Significance: Prognosis of acute myelogenous leukemia in patients over the age of 60 is poor. Recent recognition that the process of transformation utilizes a limited number of common pathways as decsribed by Hanahan and Weinberg provided the impetus to validating and extending previously published observation that immunization with normal human white blood cells (WBC) whose surface charge had been modified in vitro elicited an antibody response that cross-reacted against a broad range of leukemias (MB Sahasrabudhe Nature 1971). Specific Aim: Isolation and molecular characterization of a shared antigenic moiety from human leukemia cells. Methods: Peripheral blood was collected from healthy volunteers in ACD tubes. Red blood cells were removed by dextran sedimentation and hypotonic shock lysis. The WBCs were incubated with fluorodinitrobenzene (FDNB) at 104 molecules per cell for 12-15 minutes at room temperature. Three rabbits were immunized with FDNB-treated cells. A control rabbit was immunized with sham-treated cells. Immune sera were collected and complement was inactivated. The sera were absorbed against untreated WBCs from normal healthy donors. Absorbed immune sera were tested against leukemia cells lines (agglutination and flowcytometry) and clinical leukemia samples by Western blotting after SDS-PAGE. Antigens from whole cell lysates and membrane prep were immunoprecipitated using IgG adsorbed on protein A/G agarose beads. Liquid chromatography and mass spectrometry (LC-MS) analysis of the immunoprecipitated was performed. Results: Sera from experimental rabbits immunized with modified WBCs agglutinated leukemia cell lines (titers 1:256 to 512) whereas serum from the rabbit immunized with untreated WBCs did not. Sera from experimental rabbits also showed greater intensity of staining of leukemia cell lines by flowcytometry. Western blots of whole cell lysates and membrane preps from clinical acute myelogenous leukemia samples and normal bone marrow were probed with pre-immune serum and immune sera from the control rabbit as well as one of the experimental rabbits. Several bands recognized by the serum from experimental rabbits, immunized with FDNB-treated WBCs, but not by serum from control rabbit, immunized with sham-treated WBCs. LC-MS of the immunoprecipitated material revealed IQGAP1 and major vault transporter proteins as being differentially recognized. Conclusion and Future Directions: Immunization of rabbits with normal peripheral blood WBCs that had been incubated with nanogram quantities of FDNB elicited an antibody response that identified IQGAP1 and major vault transporter proteins as shared feature in human acute myelogenous leukemias. Investigation of the role of IQGAP1, a critical regulator of multiple cellular functions, in human acute myelogenous leukemia will be undertaken. Citation Format: Deepak M. Sahasrabudhe, Jeremy Bechelli. Ras GTPase activating like protein (IQGAP1) in human acute myelogenous leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4122. doi:10.1158/1538-7445.AM2013-4122

Mutations in the Nucleolar Phosphoprotein, Nucleophosmin, Promote the Expression of the Oncogenic Transcription Factor MEF/ELF4 in Leukemia Cells and Potentiates Transformation

Myeloid ELF1-like factor (MEF/ELF4), a member of the ETS transcription factors, can function as an oncogene in murine cancer models and is overexpressed in various human cancers. Here, we report a mechanism by which MEF/ELF4 may be activated by a common leukemia-associated mutation in the nucleophosmin gene. By using a tandem affinity purification assay, we found that MEF/ELF4 interacts with multifactorial protein nucleophosmin (NPM1). Coimmunoprecipitation and GST pull-down experiments demonstrated that MEF/ELF4 directly forms a complex with NPM1 and also identified the region of NPM1 that is responsible for this interaction. Functional analyses showed that wild-type NPM1 inhibited the DNA binding and transcriptional activity of MEF/ELF4 on the HDM2 promoter, whereas NPM1 mutant protein (Mt-NPM1) enhanced these activities of MEF/ELF4. Induction of Mt-NPM1 into MEF/ELF4-overexpressing NIH3T3 cells facilitated malignant transformation. In addition, clinical leukemia samples with NPM1 mutations had higher human MDM2 (HDM2) mRNA expression. Our data suggest that enhanced HDM2 expression induced by mutant NPM1 may have a role in MEF/ELF4-dependent leukemogenesis.

A multivalent DNA aptamer specific for the B-cell receptor on human lymphoma and leukemia

Long-term survival still eludes most patients with leukemia and non-Hodgkin’s lymphoma. No approved therapies target the hallmark of the B cell, its mIgM, also known as the B-cell receptor (BCR). Aptamers are small oligonucleotides that can specifically bind to a wide range of target molecules and offer some advantages over antibodies as therapeutic agents. Here, we report the rational engineering of aptamer TD05 into multimeric forms reactive with the BCR that may be useful in biomedical applications. Systematic truncation of TD05 coupled with modification with locked nucleic acids (LNA) increased conformational stability and nuclease resistance. Trimeric and tetrameric versions with optimized polyethyleneglycol (PEG) linker lengths exhibited high avidity at physiological temperatures both in vitro and in vivo. Competition and protease studies showed that the multimeric, optimized aptamer bound to membrane-associated human mIgM, but not with soluble IgM in plasma, allowing the possibility of targeting leukemias and lymphomas in vivo. The B-cell specificity of the multivalent aptamer was confirmed on lymphoma cell lines and fresh clinical leukemia samples. The chemically engineered aptamers, with significantly improved kinetic and biochemical features, unique specificity and desirable pharmacological properties, may be useful in biomedical applications.

Involvement of ROS in chlorogenic acid-induced apoptosis of Bcr-Abl + CML cells

Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological properties including induction of apoptosis of Bcr-Abl + chronic myeloid leukemia (CML) cell lines and clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive oxygen species (ROS) in Bcr-Abl + cells leading to downregulation of Bcr-Abl phosphorylation and apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chl-induced caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation and CML cell death. Our data suggested the involvement of parallel death pathways that converged in mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells from CML patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish the role of ROS for Chl-mediated preferential killing of Bcr-Abl + cells.

Pancratistatin induces apoptosis in clinical leukemia samples with minimal effect on non-cancerous peripheral blood mononuclear cells

BackgroundPancratistatin, a natural compound extracted from Hymenocallis littoralis, can selectively induce apoptosis in several cancer cell lines. In this ex vivo study, we evaluated the effect of pancratistatin on peripheral blood mononuclear cells obtained from 15 leukemia patients prior to clinical intervention of newly diagnosed patients, as well as others of different ages in relapse and at various disease progression states.ResultsMononuclear cells from healthy volunteers and leukemia patients were exposed to 1 μM pancratistatin for up to 48 h. Irrespective of leukemia type, pancratistatin induced apoptosis in the leukemic samples, with minimal effects on non-cancerous peripheral blood mononuclear control cells.ConclusionOur results show that pancratistatin is an effective and selective anti-cancer agent with potential for advancement to clinical trials.

Multivalent DNA Aptamer-Based Therapeutic Agents for Lymphoma and Leukemia.

Abstract 2711Poster Board II-687Non-Hodgkins lymphomas affect 450,000 patients in the United States and even with recent advances in antibody-based therapies; more than 20,000 people will die of their disease annually. The goal of this work was to develop a high affinity, stable aptamer selective for B-cell leukemias and lymphomas. Aptamers are small DNA molecules that have the ability to bind to proteins with high affinity and specificity. They are also ideal candidates as therapeutic carriers. Aptamer binding is based on the ability of small oligonucleotide polymers (typically 20–80mers) to fold into unique three-dimensional structures that can interact with a specific target. Based on nature of this interaction, aptamers could be considered to be antibody analogs. Compared to antibodies however, one of the inherent properties of aptamers is that their small size (typically 10–20,000 daltons for aptamers vs 150,000 daltons for antibodies,) might address some difficult pharmacologic issues of antibodies, which penetrate slowly into tumors and clear the blood slowly. Recently, the TDO5 aptamer was identified and it was found to bind to membrane bound human IgM, a component of the BCR complex in B-Cell neoplasms. In contrast to currently available monoclonal antibodies, TD05 binds to membrane bound human IgM only and not with soluble IgM, eliminating the possibility of competitive inhibition by soluble IgM in the serum. The specificity of the aptamer was confirmed by screening with 24 cell lines and fresh clinical leukemia samples. Out of 24 cell lines, the IgM-negative cells, including T-cell leukemias and solid tumor lines such as breast, kidney, and colon and ovarian, showed no binding with TD05 indicating there is no non-specific adhesion with cell lines. One of the challenges of using the current form of TD05 as a drug carrier is that it is not yet suitable for use in vivo because of low avidity (>10uM) and stability (t ½= 1min) at physiological conditions. In order to increase the affinity of this aptamer, a new truncated multivalent and nuclease stable aptamer was designed. First, truncation of the original version of TD05 was considered because reduced size may lead to more efficient chemical syntheses and better pharmacologic properties. The resulting TD05.1 has a 5-fold increased affinity compared to original version of TD05. Second, bivalent (BV) aptamers utilizing TD05.1 with various polyethylene glycol (PEG) linker lengths were designed. Linker length is critical in designing multivalent aptamers to avoid loss of binding due to steric hindrance and to optimize the binding geometry, both of which would affect binding affinity. An optimal linker length of ∼16nm was chosen after empiric binding studies. Nuclease stability was also addressed by the introduction of chemical functionalities into TD05.1. The introduction of non-natural bases such as LNA bases (Locked Nucleic Acids), have been demonstrated to be effective in this regard. The stability of LNA stems from the bicyclic furanose unit locked in a sugar conformation. In order to retain the specific recognition and 3-dimensional nature of the aptamer's folding, LNA analogues were only introduced at the regions that are not involved in binding. Additionally, to further improve nuclease resistance, increase circulation time in vivo and to prevent non-specific adhesion to serum proteins and cells, the LNA modified BV aptamer TD05.1 was modified with polyethylene glycol at the 3' and 5' ends. The introduction of LNA and polyethylene glycol further stabilized the secondary structure, increasing the affinity and nuclease resistance in serum from one minute to seven hours(t ½> 420 min). We then constructed a trivalent analog of the aptamer with multiple functionalities, including chelators and fluorophores, which showed ∼25-fold higher affinity compared to monomeric aptamer at physiological conditions. In conclusion, this study demonstrates the development of a trivalent, high affinity aptamer selective for the membrane bound human IgM found in B-cell leukemias and lymphoma. Studies to assess the biological activity and the use of this construct as a drug carrier to treat B-NHL and B-CLL are in progress. Disclosures:No relevant conflicts of interest to declare.

Pathogenetic Significance of Retrovirus-Tagged Mouse Myeloid Leukemia Genes for Human AML.

Acute myeloid leukemia (AML) is a heterogeneous disease with variable responses to treatment, characterized by specific chromosomal breakpoint products such as AML-ETO, PML-RAR or mutations in e.g., c/EBPα or FLT3 genes. These abnormalities are not sufficient to cause AML and approximately 40% of AML cases present without these defects or have more complex abnormalities. To find genes involved in AML, we recently isolated common integration sites (CIS) from Graffi-1.4 MuLV (Gr1.4)-induced mouse myeloid leukemia and identified the genes affected by these integrations (Erkeland S.J., et al, J. Virol., 2004). To assess the significance of these genes for human AML, we determined their contribution to 16 distinct classes of patients recently defined based on gene expression profiling of 285 AML samples using Affymetrix U133A microarrays (Valk P.J., et al, NEJM, 2004) plus 11 additional groups based on known chromosomal aberrations or gene mutations. Using the significance analysis of microarrays (SAM) methods we could link specific gene sets to these 27 patient classes. Genes were considered to contribute to the signature of the class when the following criteria were met: an RNA expression Fold Change over 1.5 or under 0.67, a score over 4 or less than −4 and a q-value less then 5%. Three gene lists derived from the Gr1.4-induced leukemia samples were generated: (I) genes (n=51, represented by 116 probe sets), within or most proximal to CIS; (II) genes (n=53, 81 probe sets) located next to the CIS flanking genes; (III) genes (n=279, 468 probe sets) located more distantly from the CIS (up to 1 megabases), with a maximum of 5 genes in each direction. Each of these gene lists was compared to the 27 outcomes of the SAM analyses to count the number of cases where a probe set of one of the three lists was significantly deregulated. To establish whether the fraction of deregulated genes in one of the three lists was significantly higher than the expected fraction of the total list, a chi-square test was performed. The list of CIS shows a significant overrepresentation of deregulated genes (Ψ2 =13.4 and p=0.0002). In contrast, the list of two closest flanking genes does not show overrepresentation (Ψ2= 0.4 and p=0.54), and neither does the list of 10 neighboring genes (Ψ2= 1.7 and p= 0.19). We repeated the analysis on a profiling dataset from 130 childhood AML samples (Ross ME, et al, Blood 2004). Using this dataset, we performed 5 SAM analyses based on known chromosomal aberrations or specific FAB-type. Again, this showed an overrepresentation of deregulated genes in case of the CIS (Ψ2= 6.3, p=0.012) but not for the more distant genes (2 closest genes: Ψ2= 0.006, p=0.936, 10 neighboring genes: Ψ2= 1.67, p=0.196). We conclude from these analyses that the closest CIS flanking genes have the highest probability to be involved in human AML. We next performed a similar analysis using the list of virus integrations from the BXH2 myeloid leukemia model (http://genome2.ncifcrf.gov/RTCGD/). This list, consisting of 53 genes represented by 111 probe sets also showed a higher representation of deregulated genes of adult AML (Ψ2= 112.2, p=0.000) as well as of pediatric AML samples (Ψ2= 14.0 and p=0.0001). Examples of CIS genes that are found associated with specific subgroups of clinical AML are CTNNA1, IRS-2, VDUP1, DUSP10 and PRDXII. These results demonstrate the power of combining retroviral tagging of cancer genes with expression array analysis of clinical leukemia samples for the identification of pathogenetic mechanisms in human AML.

Identification and characterization of nucleophosmin/B23/numatrin which binds the anti-oncogenic transcription factor IRF-1 and manifests oncogenic activity.

Interferon regulatory factor-1 (IRF-1) acts as a transcriptional activator in the interferon system and as a tumor suppressor. The loss of functional IRF-1 has been observed in a significant number of patients with myelodysplastic syndrome (MDS) and leukemia, suggesting a potentially critical role of IRF-1 in human oncostasis. Here we report an alternative mechanism by which IRF-1 may be inactivated. We purified an IRF-1 association molecule which was revealed to be identical to a nuclear factor nucleophosmin (NPM)/B23/numatrin. Functional analysis showed that NPM inhibited the DNA-binding and transcriptional activity of IRF-1. Moreover, NPM was overexpressed in several clinical leukemia samples and human-derived leukemia cell lines. Finally, overexpression of NPM in NIH3T3 cells resulted in malignant transformation. These results suggest the possible involvement of NPM in inactivating IRF-1-dependent anti-oncogenic surveillance in human cancer development.

Altered Formation of Topotecan-Stabilized Topoisomerase I-DNA Adducts in Human Leukemia Cells

AbstractTopotecan (TPT) is a topoisomerase I (topo I) poison that has shown promising antineoplastic activity in solid tumors and acute leukemia. In the present study, a band depletion assay was used to evaluate the ability of TPT to stabilize topo I-DNA adducts in human leukemia cell lines and in clinical leukemia samples ex vivo. This assay showed that 50% of the cellular topo I in HL-60 human myelomonocytic leukemia cells became covalently bound to DNA at an extracellular TPT concentration of 4 μmol/L. In contrast, in 13 clinical specimens of human leukemia harvested before treatment of patients with TPT, the TPT concentration required to stabilize 50% of the cellular topo I in topo I-DNA complexes ranged from 3 to greater than 100 μmol/L (median, 30 μmol/L). Flow microfluorimetry showed that cellular TPT accumulation varied over only a twofold range and failed to provide evidence for transport-mediated resistance in the clinical samples. These observations raise the possibility that formation of topo I-DNA adducts is diminished in many specimens of refractory/relapsed acute leukemia by a mechanism that might alter topo I sensitivity to TPT.

Toxic effects of arsenic (As3+) and other metal ions on acute promyelocytic leukemia cells.

Since arsenic compounds reportedly induced complete remission in patients with acute promyelocytic leukemia (APL) in China, we studied the in vitro effect of metal ions including As3+, As5+, Cd2+, Ga3+, Ge4+, Hg2+, Se4+, and Zn2+ on myeloid cell lines. One-tenth microM As3+ caused growth suppression and morphological changes resembling differentiation in NB4 cells, but did not induce the maturation-markers, CD11b, CD14 and NBT-reductase. More than 1 microM As3+ caused the time- and dose-dependent apoptosis of NB4 cells. Other metal ions at the same concentrations induced neither morphological changes nor apoptosis in myeloid cell lines including NB4, whereas Cd2+, Ga3+, and Hg2+ induced moderate and non-specific growth suppression. All-trans retinoic acid (ATRA)-resistant NB4 cells were similarly sensitive to As3+. Among the clinical leukemia samples, As3+ was selectively toxic to APL cells regardless of ATRA-sensitivity. These findings suggest that APL cells are sensitive to As3+, and that As3+ acts on APL cells via a different pathway than ATRA.

Molecular analysis of T cell receptor Vβ chain to detect leukemia cell clonality in patients by adaptor ligation-mediated polymerase chain reaction

We have developed a simple and rapid method to analyze the clonality of leukemia cells. After three rounds of amplification by adaptor-ligation polymerase chain reaction (PCR), the cDNA is cut with AluI, HaeIII, RsaI, and Sau3AI, and analyzed by polyacrylamide gel electrophoresis. The size of the restriction fragments is compared to that of the published restriction fragments size each TCR-β subfamily V region. The sensitivity of adaptor-ligation PCR restriction enzyme analysis (AL-PCR-REA) was 10 −4 MOLT-4 T-ALL cell population in the normal peripheral blood lymphocytes (PBL). Application of AL-PCR-REA to PBL and bone marrow (BM) cells from eight clinical leukemia samples indicated that a detection sensitivity was rather low, but revealed the clonality of all eight clinical samples. This AL-PCR-REA method can detect clonality without the need for either radioisotopes or sequencing procedures.

Detection of allelic loss within the β1-interferon gene in childhood acute lymphoblastic leukemia using differential PCR

Deletion of the short arm of chromosome 9p involving the beta 1-interferon (IFN) gene has been implicated in the process of malignant transformation in lymphomas and acute lymphoblastic leukemias. Since cytogenetic analysis is frequently unsuccessful in clinical samples, we used a recently described differential PCR technique to detect losses within the beta 1-IFN gene in 86 acute leukemias. Using differential PCR, no beta 1-IFN deletion was detected in 44 acute myeloid leukemia (AML) and eight control samples. However, five of 42 acute lymphoblastic leukemia (ALL) probes (12%) exhibited loss of the beta 1-IFN gene (three common ALL, two T-ALL). Cytogenetic analysis was performed independently in three of these five cases and revealed abnormalities of chromosome 9p in two samples. Two of five T-ALL cases exhibited a loss within the beta 1-IFN gene, compared with 3/29 c-ALLs, suggesting a predominance of IFN gene loss in T-ALLs. These data indicate that PCR can be used for rapid detection of gene dosage phenomena in clinical leukemia samples.