carbocyanines and Leukemia--Myeloid--Acute

Aptamers are short single-stranded DNA or RNA molecules, which have recently been developed for potential broad applications such as clinical therapeutics, diagnosis and tumor-targeted drug delivery. However, the selection of specific aptamers is often unsatisfactory using the classical protein or cell-based SELEX. Herein, we modified the paired cell line approach to identify aptamers targeting leukemia cells expressing the CD33 antigen. Our strategy artfully used the same cells for negative (HEK293T cells) and positive (CD33 transfected-HEK293T cells) aptamer selections, and the negative selections were performed adequately before the positive selection to remove unspecific sequences. The advantages of this strategy are that it is fast and accurate, where only a few rounds of selection together with PCR amplifications are sufficient to obtain high binding affinity antigen-targeted aptamers. By using our modified approach, we successfully obtained the CD33-targeting aptamer S30, which could highly recognize the C2 domain of the CD33 antigen in vitro and in vivo. Moreover, the optimized aptamer S30-T1 (i.e., core region of S30) was conjugated with doxorubicin (Dox) to synthesize S30-T1-Dox conjugates, which could specifically inhibit CD33 positive acute myeloid leukemia HL-60 cell proliferation by arresting the cell cycle at the G2 phase. Thus, our modified approach can rapidly screen reliable, stable and high binding affinity aptamers for precise cancer treatment.

Topics: Animals; Aptamers, Nucleotide; Carbocyanines; Cell Proliferation; DNA, Single-Stranded; Doxorubicin; G2 Phase Cell Cycle Checkpoints; HEK293 Cells; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Male; Mice; Mice, Inbred NOD; Mice, SCID; Microscopy, Confocal; Optical Imaging; Sialic Acid Binding Ig-like Lectin 3; Tissue Distribution; Transplantation, Heterologous

Elimination of leukemia stem cells (LSCs) is necessary for the destruction of malignant cell populations. Owing to the very small number of LSCs in leukemia cells, xenotransplantation studies are difficult in terms of functionally and pathophysiologically replicating clinical conditions of cell culture experiments. There is currently a limited number of lead compounds that target LSCs. Using the LSC-xenograft zebrafish screening method we previously developed, we found that the fluorescent compound 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)) selectively marked LSCs and suppressed their proliferation in vivo and in vitro. DiOC5(3) had no obvious toxicity to human umbilical cord blood CD34+ progenitor cells and normal zebrafish. It accumulated in mitochondria through organic anion transporter polypeptides that are overexpressed in the plasma membrane of LSCs, and induced apoptosis via ROS overproduction. DiOC5(3) also inhibited the nuclear translocation of NF-κB through the downregulation of LSC-selective pathways, as indicated from DNA microarray analysis. In summary, DiOC5(3) is a new type of anti-LSC compound available for diagnostic imaging and therapeutics that has the advantage of being a single fluorescent chemical.

Topics: Animals; Apoptosis; Carbocyanines; Cell Line; Cell Proliferation; Fluorescent Dyes; Humans; Leukemia, Myeloid, Acute; Mitochondria; Neoplastic Stem Cells; Reactive Oxygen Species; Zebrafish

The purpose of the study is to establish a simple and relatively inexpensive flow cytometric chemosensitivity assay (FCCA) for leukemia to distinguish leukemic blasts from normal leukocytes in clinical samples.. We first examined whether the FCCA with the mitochondrial membrane depolarization sensor, 5, 50, 6, 60-tetrachloro-1, 10, 3, 30 tetraethyl benzimidazolo carbocyanine iodide (JC-1), could detect drug-induced apoptosis as the conventional FCCA by annexin V/7-AAD detection did and whether it was applicable in the clinical samples. Second, we compared the results of the FCCA for prednisolone (PSL) with clinical PSL response in 18 acute lymphoblastic leukemia (ALL) patients to evaluate the reliability of the JC-1 FCCA. Finally, we performed the JC-1 FCCA for bortezomib (Bor) in 25 ALL or 11 acute myeloid leukemia (AML) samples as the example of the clinical application of the FCCA.. In ALL cells, the results of the JC-1 FCCA for nine anticancer drugs were well correlated with those of the conventional FCCA using anti-annexin V antibody (P < 0.001). In the clinical samples from 18 children with ALL, the results of the JC-1 FCCA for PSL were significantly correlated with the clinical PSL response (P = 0.005). In ALL samples, the sensitivity for Bor was found to be significantly correlated with the sensitivity for PSL (P = 0.005). In AML samples, the Bor sensitivity was strongly correlated with the cytarabine sensitivity (P = 0.0003).. This study showed the reliability of a relatively simple and the FCCA using JC-1, and the possibility for the further clinical application.

Topics: Adolescent; Annexin A5; Antineoplastic Agents; Apoptosis; Benzimidazoles; Carbocyanines; Cell Line, Tumor; Child; Child, Preschool; Flow Cytometry; Fluorescent Dyes; Humans; Infant; Leukemia, Myeloid, Acute; Membrane Potential, Mitochondrial; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Reproducibility of Results; Sensitivity and Specificity

The expression and activity of P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP1) were analyzed in 178 leukemia samples. Rhodamine-123 (Rho-123) and DiOC(2) were used as substrate to evaluate efflux pump activity. Chronic myeloid leukemia (CML) exhibited a higher percentage of positivity using Rho-123 than DiOC(2) (p=0.000) as compared to other types of leukemia. Moreover, Rho-123 was able to detected Pgp positive cells in a higher proportion of samples than DiOC(2) samples (p=0.004). Similarly, MRP1 positive cells were best detected by Rho-123 as opposed to DiOC(2) (p=0.003). The co-functionality of Rho-123 and DiOC(2) was observed in 26 out of 105 (24.8%) leukemic samples. Co-expression between Pgp and MRP1 was detected in 30 out of 56 (53.6%) samples. As a whole, when the same samples were analyzed, Rho-123 was able to detect Pgp positive cells in a higher proportion of samples than DiOC(2) (p=0.000). Similarly, MRP1 positive cells were best detected by Rho-123 as opposed to DiOC(2) (p=0.007). Our results support the idea that Rho-123 is the substrate of choice for leukemic cells.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Carbocyanines; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Flow Cytometry; Fluorescent Dyes; Humans; Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Multidrug Resistance-Associated Proteins; Phenotype; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Rhodamine 123; Tumor Cells, Cultured

We studied expression and functional activity of tumor cell P-gp in children with various leukemia variants and analyzed its prognostic role in acute lymphoblastic leukemia. Functional activity of P-gp increased in acute myeloid leukemia, relapses of acute lymphoblastic leukemia (in comparison with primary disease), and in a group of patients in whom no remission was attained. The survival of patients with acute lymphoblastic leukemia was lower in cases with increased expression and function of P-gp.

Topics: Adolescent; Antibodies, Monoclonal; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzimidazoles; Carbocyanines; Child; Child, Preschool; Flow Cytometry; Gene Expression; Humans; Infant; Leukemia, Myeloid, Acute; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prognosis; Statistics, Nonparametric; Survival Analysis

One of the possible causes of treatment failure in acute leukemia is the emergence of multidrug resistance caused by P-glycoprotein (P-gp) overexpression. We compared a flow cytometric assay using JC-1 with a technique using rhodamine 123 (rho123) to evaluate the P-gp function in acute leukemia. Samples from 50 acute leukemia patients were analyzed by both functional assays. The P-gp expression was assessed by an immunological flow cytometric test and the association between the P-gp status and the clinical outcome was evaluated. Of all samples, 28% showed a reversible JC-1 efflux and 36% scored positive for the rho123 assay. In two cases, the leukemic blasts showed a reversible JC-1 efflux whereas they were negative for rho123. These patients had blast cells with a very low P-gp activity. Six samples scored positive for the rho123 assay but were negative for the JC-1 test. Five of these samples did not express P-glycoprotein and were considered false positive. We found a strong correlation between the JC-1 and the rho123 test (R(s)=0.59, p<0.0001) and the JC-1 and the immunological assay (R(s)=0.29, P=0.05). There was also an association between the JC-1 status and the clinical outcome of adult patients (chi2=6.30, P=0.04). In conclusion, we recommend the JC-1 assay to study the P-gp activity in acute leukemia because it is more specific and less labor intensive than conventional functional flow cytometric tests using rhodamine 123. In addition, the JC-1 assay can be used to identify adult patients with an increased risk for adverse clinical outcome.

Topics: Adult; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzimidazoles; Carbocyanines; Child; Drug Resistance, Neoplasm; Female; Flow Cytometry; Fluorescent Dyes; Humans; K562 Cells; Leukemia; Leukemia, Myeloid, Acute; Male; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Rhodamine 123; Risk; Treatment Outcome

Nonresponse to remission-induction chemotherapy, which remains a major problem in acute myeloblastic leukemia (AML), has been linked to cellular resistance to apoptosis. Because the apoptosis induced by chemotherapeutic drugs is mediated by loss of mitochondrial transmembrane potential (MTP), it was postulated that sensitivity to mitochondrial membrane depolarization might be heterogeneous in AML. Using the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (mClCCP), the mitochondrial membrane sensitivity to depolarization (mClCCP concentrations that inhibit 50% of the transmembrane potential [IC(50)]) in AML blasts was measured and demonstrated marked interclonal heterogeneity, with the existence of comparatively sensitive (median mClCCP IC(50), 4 microM) and resistant (median mClCCP IC(50), 10 microM) clones. Furthermore, the mClCCP IC(50) was inversely associated with spontaneous in vitro apoptosis (P =.001). It was high in cases with mutant TP53 and correlated with the total cellular level of the multidrug resistance-associated protein (P =.019) but not of bcl-2, bax, or bcl-x. It was also found that the dithiol oxidant diamide, in contrast to the monovalent thiol oxidant diethyl maleate, increased the sensitivity of mitochondrial membranes to mClCCP. To confirm that TP53 directly affects MTP in leukemic cells and to establish the role of vicinal thiol oxidation in the TP53-dependent pathway, CEM 4G5 leukemia cells with forced, temperature-dependent expression of TP53 were studied. Monobromobimane, which inhibits mitochondrial membrane depolarization by preventing dithiol cross-linking, inhibited depolarization and apoptosis in 4G5 cells. It was concluded that in leukemia, TP53 and vicinal thiol/disulfide status are determinants of mitochondrial membrane sensitivity to depolarization, which is in turn associated with spontaneous apoptosis.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Disulfides; Fluorescent Dyes; Humans; Intracellular Membranes; Leukemia, Myeloid, Acute; Maleates; Membrane Potentials; Mitochondria; Mutation; Oxidation-Reduction; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfhydryl Compounds; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Uncoupling Agents