carbocyanines and Leukemia

Dielectrophoresis (DEP) can be used to noninvasively measure the dielectric state of the cell, and this data can be used to monitor cell health or apoptosis. In this study, we followed events associated with cytosine arabinoside (Ara-C)-induced apoptosis in NB4 cells using DEP analysis. Our data showed that the membrane capacitance of NB4 cells decreases from 9.42 to 7.63 mF/m(2) in the first 2 hours following treatment with Ara-C, and that this decreased capacitance persists for >12 hours. Additionally, cytoplasmic conductivity decreases from 0.217 to 0.190 S/m within 2 hours of Ara-C treatment; this level is maintained for a short period of time before decreasing. We also investigated these events molecularly at the level of gene expression using microarray analysis and showed that the expression of genes related to membrane capacitance and cytoplasmic conductivity change dramatically as early as 2 hours post-Ara-C treatment, and further demonstrated a temporal relationship between the dielectric properties and key events in apoptosis. This study, integrating physical electrical properties of the cell membrane and cytoplasm with those of conductivity-related gene networks, provides new insights into the molecular mechanisms underlying the initiation of apoptosis, establishing a systematic foundation for DEP application in follow-up drug screening and development of medicines for treating leukemia.

Topics: Annexin A5; Apoptosis; Benzimidazoles; Carbocyanines; Cell Line, Tumor; Cluster Analysis; Cytarabine; Electric Conductivity; Electrophoresis; Gene Expression Profiling; Humans; Leukemia; Microscopy, Electron, Scanning; Real-Time Polymerase Chain Reaction

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

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

Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.

Topics: Base Sequence; Biological Transport; Carbocyanines; Cell Line, Tumor; Electroporation; Flow Cytometry; Fluoresceins; Fluorescent Dyes; HL-60 Cells; Humans; Leukemia; RNA, Small Interfering; Substrate Specificity; U937 Cells

Early during apoptosis, there is a reduction in mitochondrial transmembrane potential (MTP) and externalization of phosphatidylserine (PS) in cell membrane prior to eventual cell death. Flow cytometric detection techniques targeting these changes, reduction of DiOC(6)(3) uptake upon the collapse of MTP and annexin V binding to PS have been successfully used to detect apoptotic cells. These methods have given comparable results when cell lines were used. We compared the two different techniques, DiOC(6)(3) uptake and Annexin V-propidium iodide co-labeling in the quantification of cytarabine, vincristine and daunorubicin induced apoptosis on three leukemia cell lines (HL-60, CEM, U937), and bone marrow blasts from 26 children with acute myeloid leukemia, 14 with T cell acute lymphoblastic leukemia. Anti-Fas-induced apoptosis in culture-grown peripheral blood T lymphocytes on 18 samples from 9 children with non-malignant conditions were also studied by these techniques. Our results showed that there is a correlation (P < 0. 05) between the apoptosis rates measured by these two techniques for drug-induced apoptosis in myeloid and lymphoid blasts, and for anti-Fas mAb-induced apoptosis in T lymphocytes. This data suggests that reduction of the MTP and PS externalization may be common to many apoptotic pathways and techniques targeting either of these changes may be used in quantification of apoptosis in different clinical samples.

Topics: Annexin A5; Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Carbocyanines; Child; Cytarabine; Daunorubicin; Flow Cytometry; Fluorescent Dyes; HL-60 Cells; Humans; Indicators and Reagents; Leukemia; Propidium; T-Lymphocytes; Tumor Cells, Cultured; U937 Cells; Vincristine