Rumiana Bakalova

Phenothiazines suppress proliferation and induce apoptosis in cultured leukemic cells without any influence on the viability of normal lymphocytes - Phenothiazines and leukemia

PurposeThe purpose of the present study was to investigate the effects of phenothiazines (at clinically relevant doses) on the viability and proliferation of leukemic cell lines and normal lymphocytes, and to investigate the possibility of specific induction of apoptosis in leukemic cells.MethodsPhenothiazines with different chemical structure and hydrophobicity were used: chlorpromazine (CPZ); levomepromazine (LVPZ); prometazine (PMZ); trifluoperazine (TFPZ); thioridazine (TRDZ). The leukemic cell lines used were: Daudi and Raji (derived from Burkitt’s lymphoma), K-562 (derived from myelogenous leukemia), and BALL-1, MOLT-4, HPB-ALL and CCRF-HSB-2 (derived from acute lymphoblastic leukemia). The cytotoxicity of the phenothiazines was determined by a CellTiter-Glo luminescent cell viability assay, using ATP bioluminescence as a marker of cell viability as well as a marker of mitochondrial activity. The proliferation of leukemic cells was determined using a CellTiter-AQ cell proliferation assay which is based on the reduction of a methyl-tetrazolium compound to the formazan product. Apoptosis induction was estimated using phosphatidylserine (PSer) translocation to the cell surface and DNA fragmentation as characteristics of the process.ResultsPhenothiazines (at concentrations in the range 0.1–10xa0μM) did not affect the viability of normal lymphocytes during a 24-h incubation. Moreover, about 15–20% increase in ATP bioluminescence was observed in normal cells during treatment with 40xa0μM phenothiazines. In contrast, the phenothiazines manifested strong cytotoxicity and antiproliferative activity against leukemic cells. The most powerful drugs were TFPZ and TRDZ, followed by CPZ. They showed a significant cytotoxic effect against leukemic cells even at 5–10xa0μM. The most sensitive cell lines were MOLT-4 and Raji, and the most resistant were HPB-ALL and CCRF-HSB-2. All phenothiazines induced PSer exposure on the surface of leukemic cells, but not of normal lymphocytes. TFPZ, TRDZ and CPZ also induced DNA fragmentation in almost all leukemic cell lines during a 48-h incubation. The strongest apoptotic agent was TRDZ. The apoptosis induction was not accompanied by a significant release of cytochrome c from the mitochondria into the cytoplasm of native cells. Moreover, the drugs markedly suppressed Ca2+-induced cytochrome c release in isolated mitochondria of leukemic cells.ConclusionsThe results suggest that in clinically relevant doses (up to 20xa0μM) some phenothiazines (TFPZ, TRDZ, CPZ) expressed a selective cytotoxicity and antiproliferative activity, and induced apoptosis in leukemic cells without any influence on the viability of normal lymphocytes. It is considered that the mechanism of apoptosis induction in phenothiazine-treated leukemic cells is associated with inhibition of mitochondrial DNA polymerase and decreased ATP production, which are crucial events for the viability of cancer cells.

The NK-lysin derived peptide NK-2 preferentially kills cancer cells with increased surface levels of negatively charged phosphatidylserine.

The NK‐lysin derived peptide NK‐2 is a potent antibacterial, but non‐toxic to a human keratinocyte cell line and of low hemolytic activity. Its target selectivity is based upon a strong binding preference to membranes containing anionic phospholipids, which are normally not found on the surface of human cells. Here, we analyzed the interaction of NK‐2 with normal human lymphocytes and seven different human cancer cell lines and demonstrate that some of these cells expose negatively charged surface phosphatidylserine (PS), which presumably facilitates killing of the cells by NK‐2. This is underlined by the specific intercalation of the peptide into PS‐containing liposomes analyzed by fluorescence‐resonance energy transfer spectroscopy.

A history of microarrays in biomedicine

The fundamental strategy of the current postgenomic era or the era of functional genomics is to expand the scale of biologic research from studying single genes or proteins to studying all genes or proteins simultaneously using a systematic approach. As recently developed methods for obtaining genome-wide mRNA expression data, oligonucleotide and DNA microarrays are particularly powerful in the context of knowing the entire genome sequence and can provide a global view of changes in gene expression patterns in response to physiologic alterations or manipulation of transcriptional regulators. In biomedical research, such an approach will ultimately determine biologic behavior of both normal and diseased tissues, which may provide insights into disease mechanisms and identify novel markers and candidates for diagnostic, prognostic and therapeutic intervention. However, microarray technology is still in a continuous state of evolution and development, and it may take time to implement microarrays as a routine medical device. Many limitations exist and many challenges remain to be achieved to help inclusion of microarrays in clinical medicine. In this review, a brief history of microarrays in biomedical research is provided, including experimental overview, limitations, challenges and future developments.

Fractionation of normal and leukemic T-cells by lectin-affinity column chromatography

A method for rapid fractionation of normal and leukemic T-cells (Jurkat, RPMI-8402, MOLT-4), using lectin-affinity column chromatography, is described. CNBr-activated Sepharose 6MB was used as a non-mobile phase. The gel was covalently conjugated with Dolichos biflorus agglutinin (DBA) over 24 h. The normal cells were eluted by phosphate buffered saline (Ca(2+) and Mg(2+) free), while the leukemic T-cells, interacting with DBA, were removed by N-acetyl-D-galactosamine or by low-concentrated acetic acid as a mobile phase. The cell fractions were detected spectrophotometrically at 600 nm. The rate of cell elution decreased in the order: normal>leukemic T-cells. The viability and the type of separated T-cell fractions were characterized by flow cytometry, using adequate fluorescent antibodies. The interactions between leukemic T-cells and DBA-saturated Sepharose beads were examined by fluorescent microscopy, using fluorescent isothiocyanate-DBA as a fluorescent marker.

Suppression of bcr-abl synthesis by siRNAs or tyrosine kinase activity by Glivec alters different oncogenes, apoptotic/antiapoptotic genes and cell proliferation factors (microarray study)

Short 21‐mer double‐stranded/small‐interfering RNAs (ds/siRNAs) were designed to target bcr‐abl mRNA in chronic myelogenous leukemia. The ds/siRNAs were transfected into bcr‐abl‐positive K‐562 (derived from blast crisis chronic myelogenous leukemia), using lipofectamine. Penetrating of ds/siRNAs into the cells was detected by fluorescent confocal microscopy, using fluorescein‐labeled ds/siRNAs. The cells were treated with mix of three siRNA sequences (3 × 60 nM) during 6 days with three repetitive transfections. The siRNA‐treatment was accompanied with significant reduction of bcr‐abl mRNA, p210, protein tyrosine kinase activity and cell proliferation index. Treatment of cells with Glivec (during 8 days with four repetitive doses, 180 nM single dose) resulted in analogous reduction of cell proliferation activity, stronger suppression of protein tyrosine kinase activity, and very low reduction of p210. siRNA‐mix and Glivec did not affect significantly the viability of normal lymphocytes. Microarray analysis of siRNA‐ and Glivec‐treated K‐562 cells demonstrated that both pathways of bcr‐abl suppression were accompanied with overexpression and suppression of many different oncogenes, apoptotic/antiapoptotic and cell proliferation factors. The following genes of interest were found to decrease in relatively equal degree in both siRNA‐ and Glivec‐treated cells: Bcd orf1 and orf2 proto‐oncogene, chromatin‐specific transcription elongation factor FACT 140‐kDa subunit mRNA, gene encoding splicing factor SF1, and mRNA for Tec protein tyrosine kinase. siRNA‐mix and Glivec provoked overexpression of the following common genes: c‐jun proto‐oncogene, protein kinase C‐α, pvt‐1 oncogene homologue (myc activator), interleukin‐6, 1‐8D gene from interferon‐inducible gene family, tumor necrosis factor receptor superfamily (10b), and STAT‐induced STAT inhibitor.

Cytoagglutination and cytotoxicity of Wheat Germ Agglutinin isolectins against normal lymphocytes and cultured leukemic cell lines - Relationship between structure and biological activity

The relationships between degree of lectin-cell binding, cytotoxicity and cytoagglutinating activity of three Wheat Germ Agglutinin isolectins (WGA-1, WGA-2, WGA-3) against normal lymphocytes and cultured leukemic cell lines (Jurkat, MOLT-4, Raji, Daudi, K-562) were studied. All WGA-isolectins interacted in a similar degree with normal lymphocytes, while in the case of leukemic cells, the degree of isolectin-cell binding increased in the order: WGA-1< or =WGA-3<WGA-2 at isolectin concentrations 0.5 microM and higher, and WGA-3<WGA-2< or =WGA-1 at 0.25 microM isolectin concentration. The WGA interacted in higher degree with Jurkat, Raji, Daudi and K-562, followed by MOLT-4 and normal lymphocytes. The velocity of cytoagglutination in the presence of 0.25 microM WGA-isolectins increased in the order: WGA-3<WGA-2< or =WGA-1, and was better expressed in Jurkat, Raji, Daudi and K-562, followed by MOLT-4 and normal lymphocytes. The cytotoxicity of isolectins was very well expressed against Jurkat, MOLT-4, Raji and Daudi, and less expressed against K-562 and normal lymphocytes. In the case of leukemic cells, the cytotoxic effect of WGA-isolectins increased in the order: WGA-3<WGA-2=WGA-1. A very good positive correlation was determined between velocity of cytoagglutination and degree of lectin-cell binding (r=0.77, P<0.001). A good inverse correlation was found between cytotoxicity and degree of lectin-cell binding (r=-0.34, P<0.001), and poor correlation was observed between cytotoxicity and cytoagglutinating activity of WGA-isolectins (r=0.16, P<0.01). The results suggest that the WGA-isolectins, structurally distinguishable in only several amino acid sequences, interacted in different degrees with leukemic cells and manifested different cytoagglutinating and cytotoxic activity.

Relationships between degree of binding, cytotoxicity and cytoagglutinating activity of plant-derived agglutinins in normal lymphocytes and cultured leukemic cell lines

PurposeTo clarify the relationships between the degree of lectin–cell binding, cytotoxicity and cytoagglutinating activity of plant-derived lectins in normal lymphocytes and cultured leukemic cell lines.MethodsPlant lectins with different quaternary structures and saccharide specificity were used: Dolichos biflorus agglutinin (DBA), Soybean agglutinin (SBA) and Wheat germ agglutinin (WGA). The leukemic cell lines used were: Jurkat, MOLT-4, RPMI-8402, HPB-ALL, CCR-HSB-2 and BALL-1 (derived from acute lymphoblastic leukemia); Raji and Daudi (derived from Burkitts lymphoma); K-562 (derived from myelogenous leukemia). The lectin–cell binding was detected microscopically and fluorimetrically using FITC-conjugated lectins. Cytotoxicity was estimated by the CellTiter-Glo luminescent cell viability assay, and cytoagglutinating activity by a spectrophotometric method.ResultsThe binding of DBA and SBA to normal lymphocytes was negligible, while their binding to leukemic cells increased markedly with increasing lectin concentration. Analogous results were obtained for WGA. However, it was found that WGA also interacted to a significant degree with normal lymphocytes. The degree of lectin–cell binding increased in the order: DBA<SBA<WGA. The cytoagglutinating activity and cytotoxicity of lectins increased in the same order. DBA did not exhibit a cytotoxic effect against normal or leukemic cells, and showed a poor cytoagglutinating activity only in MOLT-4, CCR-HSB-2 and BALL-1 cells. SBA exhibited poor cytotoxicity against Jurkat, RPMI-8402, HPB-ALL and CCR-HSB-2 cells, but a well-defined cytotoxicity against Raji and Daudi cells. SBA showed poor cytoagglutinating activity in leukemic cells. In contrast, WGA at concentrations higher than 0.05xa0μM showed high cytotoxicity against all leukemic cell lines tested as well as against normal lymphocytes. WGA also showed a well-expressed cytoagglutinating effect in all cell lines except normal lymphocytes. There was a moderate inverse correlation between cell viability and the velocity of cytoagglutination (r=−0.56, P<0.001), and a good correlation between cell viability and the degree of lectin–cell binding (r=−0.75, P<0.001). There was a low positive correlation between the velocity of cytoagglutination and the degree of lectin–cell binding (r=0.43, P<0.001).ConclusionThe results suggest that the lectins that bound most strongly to leukemic cells expressed higher cytotoxic and cytoagglutinating activities.

Antisense inhibition of Bcr-Abl/c-Abl synthesis promotes telomerase activity and upregulates tankyrase in human leukemia cells1

Clinical studies in chronic myelogenous leukemia demonstrate that the overexpression of Bcr‐Abl tyrosine kinase is usually accompanied by relatively low telomerase activity in the chronic phase, which reverts to a high activity in blast crisis. The present study was designed to investigate the cross‐talk between both enzymes, using Bcr‐Abl‐positive K‐562 and Bcr‐Abl‐negative Jurkat cell lines, treated with antisense oligodeoxyribonucleotides (ODNs) against Bcr‐Abl/c‐Abl mRNA. The decreased amount and enzyme activity of Bcr‐Abl/c‐Abl provoked telomerase activation in both cell lines. After short‐term treatment with anti‐Bcr‐Abl/c‐Abl ODNs (6 days), no variations in hTERT and phospho‐hTERT were detected. The decreased amount of Bcr‐Abl/c‐Abl was accompanied by: alterations in telomeric associated proteins–overexpression of tankyrase and decreased amount of TRF1/Tin2, cell growth arrest of K‐562 cells, reaching a plateau after 6 days treatment, and increased proliferating activity of Jurkat cells. No changes in telomere length were detected after short‐term treatment. In contrast, after long‐term treatment with anti‐Bcr‐Abl/c‐Abl ODNs (36 days), a significant elongation of telomeres and enhancement of hTERT were established, accompanied by an increased proliferating activity of both cell lines. These data provide evidence that the inhibition of Bcr‐Abl or c‐Abl synthesis keeps a potential to restore or induce cell proliferation through telomere lengthening control and telomerase activation.

Inhibition of bcr-abl and/or c-abl gene expression by small interfering, double-stranded RNAs: Cross-talk with cell proliferation factors and other oncogenes

Short, 21‐mer, double‐stranded/small interfering RNAs (ds/siRNAs) were designed to target bcr‐abl mRNA in chronic myelogenous leukemia (CML) with a potential also to target c‐abl mRNA.

Purification of normal lymphocytes from leukemic T-cells by lectin-affinity adsorbents - Correlation with lectin-cell binding

Utilization of leukemic T-cells from normal ones, using lectin-affinity adsorbents, is described. CNBr-activated Sepharose 6MB was covalently coupled to Soybean (SBA) or Dolichos Biflorus Agglutinins (DBA), then serves as an affinity probe for separation of leukemic T-cells from normal lymphocytes. The normal lymphocytes were removed almost completely by phosphate buffered saline (Ca(2+) and Mg(2+) free) (PBS(-)) from lectin-affinity column. More than 80% of the leukemic T-cells were retained on the lectin-affinity adsorbent, whereas another 10-15% were easily removed by PBS(-). There was a very good linear correlation between percent of cells, retained on the lectin-affinity adsorbent and percent of cells, interacting with the respective free lectin (r=0.97 for SBA, and r=0.93 for DBA). The viability of normal lymphocytes was not influenced after passing through the columns. In the case of leukemic T-cells - about 90% of the easily removed cells were dead, and another 10% were viable cells, non-interacting with DBA or SBA.