Human Chronic Myelogenous Leukemia Cell Research Articles

Cotreatment with Vorinostat (Suberoylanilide Hydroxamic Acid) Enhances Activity of Dasatinib (BMS-354825) against Imatinib Mesylate–Sensitive or Imatinib Mesylate–Resistant Chronic Myelogenous Leukemia Cells

We determined the effects of vorinostat [suberoylanilide hydroxamic acid (SAHA)] and/or dasatinib, a dual Abl/Src kinase (tyrosine kinase) inhibitor, on the cultured human (K562 and LAMA-84) or primary chronic myelogenous leukemia (CML) cells, as well as on the murine pro-B BaF3 cells with ectopic expression of the unmutated and kinase domain-mutant forms of Bcr-Abl. Following exposure to dasatinib and/or vorinostat, apoptosis, loss of clonogenic survival, as well as the activity and levels of Bcr-Abl and its downstream signaling proteins were determined. Treatment with dasatinib attenuated the levels of autophosphorylated Bcr-Abl, p-CrkL, phospho-signal transducer and activator of transcription 5 (p-STAT5), p-c-Src, and p-Lyn; inhibited the activity of Lyn and c-Src; and induced apoptosis of the cultured CML cells. Combined treatment of cultured human CML and BaF3 cells with vorinostat and dasatinib induced more apoptosis than either agent alone, as well as synergistically induced loss of clonogenic survival, which was associated with greater depletion of Bcr-Abl, p-CrkL, and p-STAT5 levels. Cotreatment with dasatinib and vorinostat also attenuated the levels of Bcr-AblE255K and Bcr-AblT315I and induced apoptosis of BaF3 cells with ectopic expression of the mutant forms of Bcr-Abl. Finally, cotreatment of the primary CML cells with vorinostat and dasatinib induced more loss of cell viability and depleted Bcr-Abl or Bcr-AblT315I, p-STAT5, and p-CrkL levels than either agent alone. As shown here, the preclinical in vitro activity of vorinostat and dasatinib against cultured and primary CML cells supports the in vivo testing of the combination in imatinib mesylate-sensitive and imatinib mesylate-resistant CML cells.

Gene therapy for BCR‐ABL+ human CML with dual phosphorylation resistant p27Kip1 and stable RNA interference using an EBV vector

BCR-ABL-mediated chronic myelogenous leukemia (CML) CD34(+) cell proliferation mostly depends on the nucleo-cytoplasmic ratio of the cyclin-dependent kinase inhibitor p27. The ubiquitin-ligase SCF(Skp2) promotes degradation of phosphorylated p27 at T187 in the nucleus, resulting in G1/S progression of the cells. On the other hand, phosphatidylinositol-3-kinase (PI3K)-directed T157 nuclear localization signal (NLS) phosphorylation results in cytoplasmic sequestration of p27, leading to abnormal integrin-mediated proliferation of CD34(+) CML cells. We demonstrate the generation of an engineered Epstein-Barr virus (EBV) vector with a BAC backbone that has the unique capacity to carry doubly modified (DM) p27 (i.e. T187A, T157A p27) along with the BCR-ABL siRNA expression construct. The HSV-tk suicide gene has also been incorporated in the same vector, which promotes apoptosis in a BCR-ABL-independent pathway. Expression of DM p27 markedly inhibits proliferation of BCR-ABL(+) primary human CML cells. Moreover, DM p27 strongly inhibits the growth of imatinib-resistant CML cells, compared to the T157A p27 (SM p27). The CML growth inhibition is found to be the result of significant G1/S arrest with concomitant increase in hypophosphorylated retinoblastoma (Rb). Moreover, the EBV vector mediated stable RNA interference induces apoptosis in K562 cells and reduces myeloid colony forming units. We therefore propose a multi-gene delivery strategy for BCR-ABL(+) CML cells by targeting not only the fusion transcript, but also the downstream signaling, to overcome drug resistance in the acute phase of CML.

Quantification of change in phosphorylation of BCR-ABL kinase and its substrates in response to Imatinib treatment in human chronic myelogenous leukemia cells

Phosphorylation by the constitutively activated BCR-ABL tyrosine kinase is associated with the pathogenesis of the human chronic myelogenous leukemia (CML). It is difficult to characterize kinase response to stimuli or drug treatment because regulatory phosphorylation events are largely transient changes affecting low abundance proteins. Stable isotope labeling with amino acids in cell culture (SILAC) has emerged as a pivotal technology for quantitative proteomics. By metabolically labeling proteins with light or heavy tyrosine, we are able to quantify the change in phosphorylation of BCR-ABL kinase and its substrates in response to drug treatment in human CML cells. In this study, we observed that BCR-ABL kinase is phosphorylated at tyrosines 393 and 644, and that SH2-domain containing inositol phosphatase (SHIP)-2 and downstream of kinase (Dok)-2 are phosphorylated at tyrosine 1135 and 299, respectively. Based on the relative intensity of isotopic peptide pairs, we demonstrate that the level of phosphorylation of BCR-ABL kinase as well as SHIP-2 and Dok-2 is reduced approximately 90% upon treatment with Imatinib, a specific inhibitor of BCR-ABL kinase. Furthermore, proteins, such as SHIP-1, SH2-containing protein (SHC) and Casitas B-lineage lymphoma proto-oncogene (CBL), are also regulated by Imatinib. These results demonstrate the simplicity and utility of SILAC as a method to quantify dynamic changes in phosphorylation at specific sites in response to stimuli or drug treatment in cell culture.

Combined effects of novel tyrosine kinase inhibitor AMN107 and histone deacetylase inhibitor LBH589 against Bcr-Abl–expressing human leukemia cells

AMN107 (Novartis Pharmaceuticals, Basel, Switzerland) has potent in vitro and in vivo activity against the unmutated and most common mutant forms of Bcr-Abl. Treatment with the histone deacetylase inhibitor LBH589 (Novartis) depletes Bcr-Abl levels. We determined the effects of AMN107 and/or LBH589 in Bcr-Abl-expressing human K562 and LAMA-84 cells, as well as in primary chronic myelogenous leukemia (CML) cells. AMN107 was more potent than imatinib mesylate (IM) in inhibiting Bcr-Abl tyrosine kinase (TK) activity and attenuating p-STAT5, p-AKT, Bcl-x(L), and c-Myc levels in K562 and LAMA-84 cells. Cotreatment with LBH589 and AMN107 exerted synergistic apoptotic effects with more attenuation of p-STAT5, p-ERK1/2, c-Myc, and Bcl-x(L) and increases in p27 and Bim levels. LBH589 attenuated Bcr-Abl levels and induced apoptosis of mouse pro-B BaF3 cells containing ectopic expression of Bcr-Abl or the IM-resistant, point-mutant Bcr-AblT315I and Bcr-AblE255K. Treatment with LBH589 also depleted Bcr-Abl levels and induced apoptosis of IM-resistant primary human CML cells, including those with expression of Bcr-AblT315I. As compared with either agent alone, cotreatment with AMN107 and LBH589 induced more loss of cell viability of primary IM-resistant CML cells. Thus, cotreatment with LBH589 and AMN107 is active against cultured or primary IM-resistant CML cells, including those with expression of Bcr-AblT315I.

Oxidative stress by ascorbate/menadione association kills K562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice

The effect of oxidative stress induced by the ascorbate/menadione-redox association was examined in K562 cells, a human erythromyeloid leukaemia cell line. Our results show that ascorbate enhances menadione redox cycling, leading to the formation of intracellular reactive oxygen species (as shown by dihydrorhodamine 123 oxidation). The incubation of cells in the presence of both ascorbate/menadione and aminotriazole, a catalase inhibitor, resulted in a strong decrease of cell survival, reinforcing the role of H 2O 2 as the main oxidizing agent killing K562 cells. This cell death was not caspase-3-dependent. Indeed, neither procaspase-3 and PARP were processed and only a weak cytochrome c release was observed. Moreover, we observed only 23% of cells with depolarized mitochondria. In ascorbate/menadione-treated cells, DNA fragmentation was observed without any sign of chromatin condensation (DAPI and TUNEL tests). The cell demise by ascorbate/menadione is consistent with a necrosis-like cell death confirmed by both cytometric profile of annexin-V/propidium iodide labeled cells and by light microscopy examination. Finally, we showed that a single i.p. administration of the association of ascorbate and menadione is able to inhibit the growth of K562 cells by about 60% (in both tumour size and volume) in an immune-deficient mice model. Taken together, these results reinforced our previous claims about a potential application of the ascorbate/menadione association in cancer therapy.

Synthesis of New Glucosylated Porphyrins Bearing an α‐d‐Linkage

This paper presents the synthesis of two new glucosyl tritolylporphyrins in which the carbohydrate moiety is connected through a carboxymethyl glycosidic α‐D‐linkage. These compounds have been obtained by reaction between porphyrins bearing an amino function with a lactone prepared from the available disaccharide isomaltulose. The photocytotoxicity of these compounds against K562 human chronic myelogenous leukemia cells has been evaluated in comparison to Photofrin II.

Synthesis and photocytotoxic activity of new chlorin–polyamine conjugates

This paper reports the synthesis of new chlorin–polyamine conjugates designed to improve the targeting of cancer cells. Photocytotoxic activity of these photosensitizers was tested against human chronic myelogenous leukemia cells (K562) and compared to the effects of Photofrin II ® and chlorin e 6.

Zinc(II) and copper(II) complexes of β-substituted hydroxylporphyrins as tumor photosensitizers

Novel photosensitizers β-(hydroquinon-2-yl)-5,10,15,20-tetra(4-hydroxylphenyl)porphyrinato zinc(II) (Zn(II)P) and β-(hydroquinon-2-yl)-5,10,15,20-tetra(4-hydroxylphenyl)porphyrinato copper(II) (Cu(II)P) were synthesized and characterized. Their ability of producing singlet oxygen under irradiation was detected by the measurement of decomposition of 1,3-diphenylisobenzofuran (DPBF). The preliminary biological activity studies show that the Zn(II)P has photo-toxicity on human chronic myelogenous leukemia cell (K562) and could cleave supercoiled DNA (pBR 322 DNA), while the Cu(II)P has inferior biological activity. Results showed Zn(II)P having high anti-tumor activity, which presents a promising photosensitizer for photodynamic therapy.

BCR-ABL nuclear entrapment kills human CML cells: ex vivo study on 35 patients with the combination of imatinib mesylate and leptomycin B

The BCR-ABL oncoprotein of chronic myelogenous leukemia (CML) localizes to the cell cytoplasm, where it activates proliferative and antiapoptotic signaling pathways. We previously reported that the combination of the ABL kinase inhibitor imatinib mesylate (IM) and the nuclear export inhibitor leptomycin B (LMB) traps BCR-ABL inside the nucleus, triggering the death of the leukemic cells. To evaluate the efficacy of the combination of IM and LMB on human cells we collected CD34-positive cells from 6 healthy donors and myeloid progenitors from 35 patients with CML. The sequential addition of IM and LMB generated the strongest reduction in the proliferative potential of the leukemic cells, with limited toxicity to normal myeloid precursors. Furthermore, nested reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on colonies representative of each experimental condition demonstrated that the combination of IM and LMB was the most effective regimen in reducing the number of BCR-ABL-positive colonies. The efficacy of the 2-drug association was independent of the clinical characteristics of the patients. Our results indicate that strategies aimed at the nuclear entrapment of BCR-ABL efficiently kill human leukemic cells, suggesting that the clinical development of this approach could be of significant therapeutic value for newly diagnosed and IM-resistant CML patients.

Analysis of Membrane Proteins from Human Chronic Myelogenous Leukemia Cells: Comparison of Extraction Methods for Multidimensional LC−MS/MS

An important strategy for "shotgun proteomics" profiling involves solution proteolysis of proteins, followed by peptide separation using multidimensional liquid chromatography and automated sequencing by mass spectrometry (LC-MS/MS). Several protocols for extracting and handling membrane proteins for shotgun proteomics experiments have been reported, but few direct comparisons of different protocols have been reported. We compare four methods for preparing membrane proteins from human cells, using acid labile surfactants (ALS), urea, and mixed organic-aqueous solvents. These methods were compared with respect to their efficiency of protein solubilization and proteolysis, peptide and protein recovery, membrane protein enrichment, and peptide coverage of transmembrane proteins. Overall, approximately 50-60% of proteins recovered were membrane-associated, identified from Gene Ontology annotations and transmembrane prediction software. Samples extracted with ALS, extracted with urea followed by dilution, or extracted with urea followed by desalting yielded comparable peptide recoveries and sequence coverage of transmembrane proteins. In contrast, suboptimal proteolysis was observed with organic solvent. Urea extraction followed by desalting may be a particularly useful approach, as it is less costly than ALS and yields satisfactory protein denaturation and proteolysis under conditions that minimize reactivity with urea-derived cyanate. Spectral counting was used to compare datasets of proteins from membrane samples with those of soluble proteins from K562 cells, and to estimate fold differences in protein abundances. Proteins most highly abundant in the membrane samples showed enrichment of integral membrane protein identifications, consistent with their isolation by differential centrifugation.

677. Generation and Application of an Efficient Chronic Myelogenous Leukemia (CML)-Targeted Recombinant Adeno-Associated Virus (rAAV) Vector: New Therapeutic Options?

Top of pageAbstract Introduction: Despite great advances in vector development, most standard rAAV-2 vectors still lack the target cell specificity required for an efficient in vivo application. In addition, the gene transfer efficiency in many promising target cells (e.g.: peripheral blood progenitor cells (PBPC)) is low. Thus, designing gene therapy protocols for diseases like chronic myelogenous leukemia (CML) remained difficult, as standard rAAV-2-based vectors lack both the required gene transfer efficiency and selectivity. A recent advancement in vector development (Muller et al., 2003) now allows the generation of rAAV capsid mutants that offers higher target cell specificity and transduction efficiency. This method was applied to obtain a CML-targeted rAAV vector and was tested on a panel of leukemic cell lines, as well on primary CML material. Material and Methods: Performing latter method on the CML cell line K562 (4 selection rounds), several mutant CML-targeted rAAV2 capsid clones were obtained, cloned into an rAAV2 helper plasmid and for each of the mutants rAAV2-eGFP vector stocks were produced. Titers were verified using our real time PCR-based titration assay. To determine efficiency and specificity, a panel of leukemic (CML and AML) and non-leukemic cell lines, as well primary human CML and PBPC cells were transduced with these vectors. Mock, normal rAAV2 and random mutant clone-transduced cells served as controls. Transduction of primary human CML and PBPC cells was confirmed using FACS-sorted GFP+ cells with subsequent bcr-abl-FISH or FACS analysis for GFP/CD34+ co- expression, respectively. Results: Transduction of a panel of six leukemic cell lines with the CML-targeted rAAV2 vectors (moi 100 iu) resulted in an over 10-fold increase of gene transfer efficiency compared to random mutant and standard rAAV2 vectors; gene transfer rates of >60% were obtained. In primary human cells, for both CML (n=4) and PBPC (n=2), an increase in gene transfer was observed (CML: AAV2: 2.4 |[plusmn]| 0.2 vs AAV2-cml: 5.1 |[plusmn]| 1.6 (p=0.04). CD34+: AAV2: 3.2 |[plusmn]| 0.9 vs AAV2-cml: 8.5 |[plusmn]| 4.3 (p>0.05)). All transduced cell were positive for either the BCR-ABL fusion gene (CML) or the PBPC marker CD34. In contrast, gene transfer of the mutant CML-targeted rAAV2 vectors into non-leukemic cell lines resulted in a reduction in gene transfer efficiency compared to standard rAAV2 vectors (<50%). Conclusion: In this study, we were able to generate a CML-targeted rAAV2 vector, which was able to transduce leukemic cell lines and primary human CML cells with significant higher efficiency and specificity than standard rAAV2 vectors. The development of these vectors holds promise for future clinical applications.

Imatinib mesylate (STI571) abrogates the resistance to doxorubicin in human K562 chronic myeloid leukemia cells by inhibition of BCR/ABL kinase-mediated DNA repair

Imatinib mesylate (STI571), a specific inhibitor of BCR/ABL tyrosine kinase, exhibits potent antileukemic effects in the treatment of chronic myelogenous leukemia (CML). However, the precise mechanism by which inhibition of BCR/ABL activity results in pharmacological responses remains unknown. BCR/ABL-positive human K562 CML cells resistant to doxorubicin (K562DoxR) and their sensitive counterparts (K562DoxS) were used to determine the mechanism by which the STI571 inhibitor may overcome drug resistance. K562 wild type cells and CCRF-CEM lymphoblastic leukemia cells without BCR/ABL were used as controls. The STI571 specificity was examined by use of murine pro-B lymphoid Baf3 cells with or without BCR/ABL kinase expression. We examined kinetics of DNA repair after cell treatment with doxorubicin in the presence or absence of STI571 by the alkaline comet assay. The MTT assay was used to estimate resistance against doxorubicin and Western blot analysis with Crk-L antibody was performed to evaluate BCR/ABL kinase inhibition by STI571. We provide evidence that treatment of CML-derived BCR/ABL-expressing leukemia K562 cells with STI571 results in the inhibition of DNA repair and abrogation of the resistance of these cells to doxorubicin. We found that doxorubicin-resistant K562DoxR cells exhibited accelerated kinetics of DNA repair compared with doxorubicin-sensitive K562DoxS cells. Inhibition of BCR/ABL kinase in K562DoxR cells with 1 μM STI571 decreased the kinetics of DNA repair and abrogated drug resistance. The results suggest that STI571-mediated inhibition of BCR/ABL kinase activity can affect the effectiveness of the DNA-repair pathways, which in turn may enhance drug sensitivity of leukemia cells.

Involvement of the Drug Transporters P Glycoprotein and Multidrug Resistance-Associated Protein Mrp2 in Telithromycin Transport

The present study aims to investigate the role of P glycoprotein and multidrug resistance-associated protein (Mrp2) in the transport of telithromycin, a newly developed ketolide antibiotic, in vitro and in vivo. The in vitro experiments revealed that the intracellular accumulation of telithromycin in adriamycin-resistant human chronic myelogenous leukemia cells (K562/ADR) overexpressing P glycoprotein was significantly lower than that in human chronic myelogenous leukemia cells (K562/S) not expressing P glycoprotein. Cyclosporine significantly increased the intracellular accumulation of telithromycin in K562/ADR cells. When telithromycin was coadministered intravenously with cyclosporine in Sprague-Dawley (SD) rats, cyclosporine significantly delayed the disappearance of telithromycin from plasma and decreased its systemic clearance to 60% of the corresponding control values. Hepatobiliary excretion experiments revealed that cyclosporine almost completely inhibited the biliary clearance of telithromycin, suggesting that telithromycin is a substrate of P glycoprotein and a potential substrate of Mrp2. Moreover, the biliary clearance of telithromycin was significantly decreased by 80% in Eisai hyperbilirubinemic mutant rats with a hereditary deficiency in Mrp2, indicating that Mrp2, as well as P glycoprotein, plays an important role in the biliary excretion of telithromycin. When the effect of telithromycin on the biliary excretion of doxorubicin, a substrate of P glycoprotein and Mrp2, was examined in SD rats, telithromycin significantly decreased the biliary clearance of doxorubicin by 80%. Results obtained from this study indicate that telithromycin is a substrate of both P glycoprotein and Mrp2, and these transporters are involved in the hepatobiliary transport of telithromycin.

Activation of a Novel Proteasomal Independent Bcr/Abl Degradation Pathway by WP1130 Induces Apoptosis in CML Cells.

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells caused by 9:22 reciprocal chromosomal translocation resulting in expression of a highly stable, constitutively active tyrosine kinase, Bcr/Abl. Inhibition of Bcr/Abl with imatinib mesylate, a potent Abl-specific tyrosine kinase inhibitor, is a highly effective therapy for this disease. However, clinical resistance occurs particularly in the later stages of the disease due mainly to the occurrence of point mutations in the Abl kinase domain and continual Bcr/Abl signaling. Alternative and supplemental therapies are still needed and of great clinical interest. Screening a library of small molecular weight compounds for their ability to induce the degradation of critical transcription factors and tyrosine kinases led to the design and synthesis of WP1130, a compound that reduces Jak2 and Bcr/Abl protein stability. Treatment with WP1130 caused the rapid destruction of Bcr/Abl in human CML cell lines (K562, BV173) as well as in clinical CML specimens obtained from patients. This treatment also resulted in dephosphorylation of pSTAT5, pHck and pCrkL, three known targets of Bcr/Abl. Loss of Bcr/Abl protein following WP1130 treatment was similar to that caused by Bcr/Abl silencing (siRNA), and both treatments lead to the induction of apoptosis. Treatment with WP1130 caused rapid degradation of both wild-type and mutant (T315I) Bcr/Abl protein in BaF3 cell expressing these proteins and in a CML cell line expressing wildtype (BV173) and T315I mutant BCR-ABL (BV-173R). Treatment with WP1130 also strongly inhibited colony formation in soft agar of human CML cells from imatinib resistant patients expressing the T315I mutation. The degradation of Bcr/Abl in cells treated with WP1130 was rapid, (1h) and independent of the proteasomal system and HSP90, a chaperone associated with Bcr/Abl stability. Interestingly, WP1130-induced Bcr/Abl degradation was blocked in the presence of vanadate, a tyrosine phosphatase inhibitor. Together, these observations suggest that WP1130-induced Bcr/Abl degradation is mediated by a unique, proteasomal independent pathway in CML cells. Further development of WP1130 is underway with the goal of developing an effective therapy for treating CML by targeting Bcr/Abl protein expressed in early CML progenitors or stem cell populations.

Imatinib Mesylate (STI571) Abrogates the Resistance to Doxorubicin in K562 Chronic Myeloid Leukemia Cells by Inhibition of BCR/ABL Kinase-Mediated DNA Repair.

Imatinib mesylate (STI571), a specific inhibitor of the BCR/ABL tyrosine kinase, exhibits potent antileukemic effects in the treatment of chronic myelogenous leukemia (CML). However, the precise mechanisms by which inhibition of BCR/ABL activity results in pharmacological responses remain unknown. BCR/ABL-positive human CML cells resistant to doxorubicin K562doxoR and their sensitive K562doxoS counterparts were used to determine the mechanism by which the STI571 inhibitor may overcome drug resistance. K562 wild type cells and CCRF-CEM lymphoblastic leukemia cells without BCR/ABL were used as controls. We examined kinetics of DNA repair after cell treatment with the drug by the alkaline comet assay. MTT assay was used to estimate resistance against doxorubicin and Western Blot analysis with Crk-L antibody was performed to evaluate BCR/ABL kinase inhibition by STI571. We provide evidence that treatment of CML-derived BCR/ABL-expressing leukemia K562 cells with STI571 results in the inhibition of DNA repair and abrogation of the resistance of these cells to doxorubicin. We found that doxorubicin-resistant K562doxoR cells exhibited accelerated kinetics of DNA repair in comparison to doxorubicin-sensitive K562doxoS cells. Inhibition of BCR/ABL kinase in K562doxoR cells with 1 μM STI571 decreased the kinetics of DNA repair and abrogated drug resistance. The results suggest that STI571-mediated inhibition of BCR/ABL kinase activity can affect the effectiveness of the DNA repair pathways, which in turn may enhance drug sensitivity of leukemia cells.

Polyamine conjugates of meso-tritolylporphyrin and protoporphyrin IX: Potential agents for photodynamic therapy of cancers

An efficient five-step synthesis method was developed to obtain tritolylporphyrin and protoporphyrin IX polyamine conjugates. These compounds were composed of either one polyamine unit (spermidine or spermine) covalently tethered to monocarboxyphenyl tritolylporphyrin or two molecules of polyamines borne by protoporphyrin IX. In each compound, an aliphatic spacer arm is linked to the N 4 polyamine position. Photocytotoxicity of these new compounds was evaluated against K562 human chronic myelogenous leukemia cells and compared to Photofrin II ®; protoporphyrin IX polyamine conjugates exhibited much stronger photocytocicity than Photofrin II ® and were shown to readily induce necrosis in treated cells.

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside Inhibits Cancer Cell Proliferation in Vitro and in Vivo via AMP-activated Protein Kinase

5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is widely used as an AMP-kinase activator, which regulates energy homeostasis and response to metabolic stress. Here, we investigated the effect of AICAR, an AMPK activator, on proliferation of various cancer cells and observed that proliferation of all the examined cell lines was significantly inhibited by AICAR treatment due to arrest in S-phase accompanied with increased expression of p21, p27, and p53 proteins and inhibition of PI3K-Akt pathway. Inhibition in in vitro growth of cancer cells was mirrored in vivo with increased expression of p21, p27, and p53 and attenuation of Akt phosphorylation. Anti-proliferative effect of AICAR is mediated through activated AMP-activated protein kinase (AMPK) as iodotubericidin and dominant-negative AMPK expression vector reversed the AICAR-mediated growth arrest. Moreover, constitutive active AMPK arrested the cells in S-phase by inducing the expression of p21, p27, and p53 proteins and inhibiting Akt phosphorylation, suggesting the involvement of AMPK. AICAR inhibited proliferation in both LKB and LKB knock-out mouse embryo fibroblasts to similar extent and arrested cells at S-phase when transfected with dominant negative expression vector of LKB. Altogether, these results indicate that AICAR can be utilized as a therapeutic drug to inhibit cancer, and AMPK can be a potential target for treatment of various cancers independent of the functional tumor suppressor gene, LKB.

Effects of prolactin and cortisol on natural killer (NK) cell surface expression and function of human natural cytotoxicity receptors (NKp46, NKp44 and NKp30)

The surface density of the triggering receptors (e.g. NKp46 and NKp30) responsible for natural killer (NK) cell-mediated cytotoxicity determines the ability of NK cells to kill susceptible target cells. In this study, we show that prolactin up-regulates and cortisol down-regulates the surface expression of NKp46 and NKp30. The prolactin-mediated activation and the cortisol-mediated inhibition of natural cytotoxicity receptor (NCR) surface expression reflects gene regulation at the transcriptional level. NKp46 and NKp30 are the major receptors involved in the NK-mediated killing of K562, a human chronic myelogenous leukaemia cell line. Accordingly, the prolactin dramatically increased the NK-mediated killing of the K562 cell line, whereas cortisol abolished this activity. Our data suggest a mechanism by which prolactin activates the lytic function of NK cells, and cortisol inhibits the NK-mediated attack.

Betulinic acid induces apoptosis in human chronic myelogenous leukemia (CML) cell line K-562 without altering the levels of Bcr-Abl

Betulinic acid (BA), a plant derived triterpenoid, isolated from various sources shows cytotoxicity in cell lines of melanoma, neuroectodermal and malignant brain tumors. Chronic myelogenous leukemia (CML) is characterized by Philadelphia chromosome (Bcr-Abl), a molecular abnormality leading to the intrinsic tyrosine kinase activity that provides growth and survival advantage to the cells. Present study describes the cytotoxicity of BA on human CML cell line K-562, positive for Bcr-Abl. The decrease in the viability of K-562 cells treated with BA at different concentrations and time intervals was assessed using MTT assay. Cell death induced by BA was determined to be apoptotic as measured by FACS analysis of PI stained nuclei, PS externalization by Annexin-V fluorescence and characteristic DNA fragmentation. DiOC 6(3) fluorescent probe determined alterations in the mitochondrial membrane potential (MMP). RT-PCR confirmed the expression levels of Bcr-Abl in controls and K-562 cells treated with BA. The rapid loss of MMP of K-562 cells upon treatment with BA shows the direct activation of apoptosis at the level of mitochondria, overcoming the resistance of the high levels of expression of Bcr-Abl.

Transduction of TAT-NF-YA Fusion Protein into K562 Human Chronic Myelogenous Leukemia Cells Upregulates HOXB4 Activity.

Protein transduction to redirect hematopoietic stem cell (HSC) fate for clinical therapy is an attractive alternative to genetic modification, in that the stem cell genome is not permanently modified. Indeed, published studies suggest that protein transduction with HOXB4, similar to retroviral overexpression of HoxB4, can increase HSC competitive repopulating ability. We have recently found that the trimeric transcription factor NF-Y, by modulation of its regulated subunit NF-YA, controls the expression of several HSC activating genes, including the Hox4 paralogs HoxB4, HoxC4, HoxD4 as well as p27, telomerase, Notch1, and LEF1. We have therefore now asked whether we can deliver NF-YA to hematopoietic cells by fusion with the protein transduction domain of the HIV-1 TAT protein, and modeled the molecular and biological activity of this protein by its effects on the HoxB4 promoter in vitro and in vivo. First, we designed and tested three fusion constructs expressing human NF-YA: with an N-terminal 6-histidine leader, a C-terminal 6xHis tag, and a GST tag at the N-terminus of NF-YA. Recombinant proteins bearing either the N- or C-terminal His-tag were poorly expressed under diverse growing and induction conditions, and were highly contaminated with bacterial host proteins. GST-fusion protein showed a much higher expression level and no host protein contamination, so all experiments were performed using GST-constructs. Next, electrophoretic mobility shift assays (EMSA) demonstrated that purified GST-TAT-NF-YA binds to HoxB4 promoter probes identically to endogenously translated NF-YA. NIH 3T3 fibroblasts, or K562 cells cultured in 0.1–2mM GST-TAT-NF-YA for 1.5h, but not without recombinant protein, showed bright cytoplasmic and nuclear fluorescence upon staining with anti-GST. To test the ability of GST-TAT-NF-YA to activate the HoxB4 promoter in live cells, GST-TAT-NF-YA's effect was measured both on transfected HOXB4 promoter reporter constructs and on endogenous HoxB4 mRNA. First, when GST-TAT-NF-YA was incubated with K562 cells electroporated with HoxB4 promoter/luciferase, along with plasmids encoding the NF-Ya partners NF-YB, NF-YC, USF1, USF2 (and Renilla luciferase for transfection control), normalized luciferase activity calculated as a % relative to control HoxB4-luc activity increased in an NF-YA concentration-dependent fashion. More pronounced effects were seen when GST-TAT-NF-YA protein was added to stably transfected HOXB4-luc K562 cell lines, with up to 2.7 fold increased HoxB4-luc activity versus control cells. Importantly, addition of GST-TAT-NF-YA protein to unmanipulated K562 cells increased expression of endogenous HoxB4 mRNA 2-2.5X, as measured by real-time PCR. Taken together, these data demonstrate that NF-YA protein can be biochemically, efficiently and functionally delivered to hematopoietic cells via TAT-mediated technology, providing a promising alternative to gene transfer to stimulate multiple HSC-activating genes.