[Effect of Rapamycin on Expression of Survivin and Caspase-3 and Its Influence on K562 Cell Ultrastructure].

OBJECTIVE: To investigate the effect of rapamycin on the expression of survivin and caspase-3 at mRNA level in K562 cells and the influence of rapamycin on K562 cell ultrastructure. METHODS: The effects of rapamycin at various concentration on K562 cell proliferation were analyzed by CCK8; the morphological characteristics of K562 cells was observed by transmission electron microscopy; the expression of survivin and caspase-3 at mRNA level in K562 cells treated with rapamycin was detected by RT-PCR. RESULTS: The proliferation of K562 cells was significantly inhibited by rapamycin. The apoptosis level of K562 cells increased with increase of rapamycin concentration, the expression of survivin at mRNA level decreased with increase of rapamycin concentration (P < 0.05). The expression of caspase-3 at mRNA level increased with increase of rapamycin concentration. CONCLUSION: Rapamycin can prornote K562 cell apoptosis through up-regulating caspase-3 level and reduceing survivin level.

Apoptosis induction of K562 cells by lymphocytes: an AFM study.

Antitumor immunotherapies, as a prospective approach for local cancer treatment, are attracting increasing interests. To detect the reacting course of immune and tumor cells, we have observed the process of K562 cells (a human erythroleukemic cell line) coculturing with peripheral lymphocytes, and the morphological and ultrastructural alterations of K562 cells and lymphocytes were investigated as well using atomic force microscopy (AFM). AFM morphological imaging revealed that after coculture the apoptosis-like structures such as blebbing, pores, and apoptotic bodies were observed on the K562 cells. Also, the cell-surface roughness decreased significantly, which implied the changes in chemical composition of cell membranes. Moreover, the lymphocytes were damaged to some extent induced by the coculture. The data demonstrated that K562 cells could be attacked and induced apoptosis by lymphocytes, and they would make damages to lymphocytes to escape the surveillance of immune system.

The Wnt5a/Ror2 noncanonical signaling pathway inhibits canonical Wnt signaling in K562 cells.

Wnt5a has been shown to be involved in cancer progression in a variety of tumor types, and regulates multiple intracellular signaling cascades; it is a representative ligand that activates a noncanonical Wnt signaling pathway. The mechanism governing how Wnt5a determines the specificity of these pathways and the relationship with tumorigenesis is still unknown. In this study, we aimed to clarify the tumor suppressor role of Wnt5a in leukemogenesis. In particular, we focused on Ror2 functioning as a Wnt5a receptor to mediate noncanonical Wnt signaling, which inhibits canonical Wnt signaling in K562 cells. We found that up-regulation of Wnt5a expression increased Ror2 expression in K562 cells and Wnt5a and Ror2 were co-expressed in the cytoplasm. Also, Wnt5a induced the intrnalization of Ror2. Co-immunoprecipitation experiments were performed to determine whether Ror2 binds to Wnt5a, and inhibits Wnt5a binding with Frizzled4 and LRP5 in Wnt5a treated K562 cells. Wnt5a had no effect on total ß-catenin expression levels, but regulated tyrosine phosphorylation of ß-catenin and translocation of ß-catenin from the cytoplasm to the nucleus. Furthermore, expression of Wnt5a was associated with suppression of ß-catenin/TCF-dependent transcriptional activity and down-regulated the expression of cyclin D1, a downstream target gene of the canonical Wnt signaling pathway. We hypothesize that Wnt5a plays the role of a tumor suppressor in leukemogenesis through the Wnt5a/Ror2 noncanonical signaling pathway that inhibits Wnt canonical signaling.

Biocompatible conductive architecture of carbon nanofiber-doped chitosan prepared with controllable electrodeposition for cytosensing.

A novel architecture was designed by combining the biocompatibility of chitosan (CS) and excellent conductivity of carbon nanofiber (CNF). The controllable electrodeposition of soluble CNF-doped CS colloidal solution formed a robust CNF-CS nanocomposite film with good biocompatibility for the immobilization and cytosensing of K562 cells on an electrode. The formed architecture was characterized using scanning electron microscopic, infrared spectrum, contact angle, and thermogravimetric analyses. The adhesion of K562 cells on the nanocomposite film-modified electrode could be followed with electrochemical impedance spectroscopy and cyclic voltammetry. The presence of CNF facilitated the electrochemical behavior of K562 cells. The impedance of electronic transduction was related to the amount of the adhered cells, producing a highly sensitive impedance sensor for K562 cells ranging from 5 x 10(3) to 5.0 x 10(7) cells mL-1 with a limit of detection of 1 x 10(3) cells mL-1. This work suggested a strategy to prepare a biocompatible and conductive interface for immobilization and electrochemical detection of cells and opened a way for the application of CNF in cytosensing.

Betanin a betacyanin pigment purified from fruits of Opuntia ficus-indica induces apoptosis in human chronic myeloid leukemia Cell line-K562.

Betalains are water-soluble nitrogenous vacuolar pigments present in flowers and fruits of many caryophyllales with potent antioxidant properties. In the present study the antiproliferative effects of betanin, a principle betacyanin pigment, isolated from the fruits of Opuntia ficus-indica, was evaluated on human chronic myeloid leukemia cell line (K562). The results show dose and time dependent decrease in the proliferation of K562 cells treated with betanin with an IC(50) of 40 microM. Further studies involving scanning and transmission electron microscopy revealed the apoptotic characteristics such as chromatin condensation, cell shrinkage and membrane blebbing. Agarose electrophoresis of genomic DNA of cells treated with betanin showed fragmentation pattern typical for apoptotic cells. Flow cytometric analysis of cells treated with 40 microM betanin showed 28.4% of cells in sub G0/G1 phase. Betanin treatment to the cells also induced the release of cytochrome c into the cytosol, poly (ADP) ribose polymerase (PARP) cleavage, down regulation Bcl-2, and reduction in the membrane potentials. Confocal microscopic studies on the cells treated with betanin suggest the entry of betanin into the cells. These studies thus demonstrate that betanin induces apoptosis in K562 cells through the intrinsic pathway and is mediated by the release of cytochrome c from mitochondria into the cytosol, and PARP cleavage. The antiproliferative effects of betanin add further value to the nutritional characteristics of the fruits of O. ficus-indica.

Effects of permeability transition inhibition and decrease in cytochrome c content on doxorubicin toxicity in K562 cells.

As mitochondria play a key role in the commitment to cell death, we have investigated the mitochondrial consequences of resistance to doxorubicin (DOX) in K562 cells. We found that the permeability transition pore (PTP) inhibitor cyclosporine A (CsA) failed to inhibit PTP opening in the resistant clone. Moreover, the Ca2+ loading capacity in the resistant clone was identical to that observed in the parent cells in the presence of CsA, suggesting that the PTP was already inhibited in a CsA-like manner in the resistant cells. In agreement with this proposal, the mitochondrial target of CsA cyclophilin D (CyD) decreased by half in the resistant cells. The levels of adenine nucleotide translocator, voltage anion-dependent channel, Bax, Bcl-2, Bcl-xL, AIF and Smac/Diablo, were similar in both cell lines, whereas cytochrome c content was divided by three in the resistant cells. Since P-glycoprotein inhibition did not restore DOX toxicity in the resistant cells, while DOX-induced cell death in the parent cells was prevented by either PTP inhibition or siRNA-induced decrease in cytochrome c content, we conclude that the inhibition of PTP opening and the decrease in cytochrome c content participate in the mechanism that makes K562 cells resistant to DOX.

Oridonin-induced apoptosis in leukemia K562 cells and its mechanism.

Oridonin, an extract from the Chinese herb Rabdosia rubescens, is currently one of the most important traditional Chinese herbal medicines. Recently oridonin has been reported to have anti- tumor effects in a large variety of malignant diseases. In this study, we investigated the apoptotic inducing effect of oridonin in leukemia K562 cells and its mechanism. Cell growth inhibition was measured using a microculture tetrazolium assay, apoptosis was measured by flow cytometry and electron microscopy as well as by DNA fragmentation analysis. Telomerase activity was measured by TRAP-enzyme- linked immunosorbent assay, and the expression of Bcl-2 and Bax proteins was detected by western blot analysis. The results showed that oridonin could inhibit the proliferation and induce apoptosis on leukemia K562 cells remarkably. Telomerase activity as well as Bcl-2 expression was down- regulated, while Bax expression was up-regulated concurrently, when apoptosis ocurred. We therefore conclude that oridonin demonstrated anti-proliferative and apoptosis-inducing effects on K562 cells in vitro, and that changes in bcl-2 and bax protein levels as well as telomerase activity may play an important role in its mechanism of action.

Spectral evaluation of laser-induced cell damage with photothermal microscopy.

BACKGROUND AND OBJECTIVES: Determining cell photo-damage is important for laser medicine and laser safety standards. This work evaluated the potential of photothermal (PT) technique for studying invasive laser-cell interaction, with a focus on PT evaluation of spectral dependence of laser-induced damage in visible region at single intact cell level. STUDY DESIGN/MATERIALS AND METHODS: PT is based on irradiation of a single intact cells with a tunable pump laser pulse (420-570 nm, 8 nanoseconds, 0.1-300 microJ) and monitoring of temperature-dependent variations of the refractive index with a second, collinear probe beam in pulse (imaging) mode (639 nm, 13 nanoseconds, 10 nJ), and continuous (integrated PT response) mode (633 nm, 2 mW). The local and the integrated PT responses from the individual living red blood cells, lymphocytes, and cancer cells (K562) in vitro were obtained at different pump laser fluence and wavelength and compared with data obtained by conventional viability tests (Annexin V--propidium iodide). RESULTS: The cell damage with pump pulse lead to specific change in PT response's temporal shape and PT image's structure. The photodamage thresholds varied in the range of 0.5-5 J/cm2 for red blood cells, 4.4-42 J/cm2 for lymphocytes, and 36-90 J/cm2 for blast cells in the pump wavelength range of 417-555 nm. CONCLUSION: Damage threshold at different wavelength depends on absorption spectra of cells. Spectral evaluation of laser-damage thresholds can be done in two supplements for each PT mode--PT imaging and integrated PT response. The correlation between specific change of PT parameters and cell damage permits using PT technique to rapidly estimate the invasive conditions of the laser-cell interactions.

Heterotypic interactions between transferrin receptor and transferrin receptor 2.

Cellular iron uptake in most tissues occurs via endocytosis of diferric transferrin (Tf) bound to the transferrin receptor (TfR). Recently, a second transferrin receptor, transferrin receptor 2 (TfR2), has been identified and shown to play a critical role in iron metabolism. TfR2 is capable of Tf-mediated iron uptake and mutations in this gene result in a rare form of hereditary hemochromatosis unrelated to the hereditary hemochromatosis protein, HFE. Unlike TfR, TfR2 expression is not controlled by cellular iron concentrations and little information is currently available regarding the role of TfR2 in cellular iron homeostasis. To investigate the relationship between TfR and TfR2, we performed a series of in vivo and in vitro experiments using antibodies generated to each receptor. Western blots demonstrate that TfR2 protein is expressed strongest in erythroid/myeloid cell lines. Metabolic labeling studies indicate that TfR2 protein levels are approximately 20-fold lower than TfR in these cells. TfR and TfR2 have similar cellular localizations in K562 cells and coimmunoprecipitate to only a very limited extent. Western analysis of the receptors under nonreducing conditions reveals that they can form heterodimers.

Distribution of actin in etoposide-induced human leukemia cell line K-562 using fluorescence and immunoelectron microscopy technique.

Localization of actin was studied in erythroleukemic cell line K-562 after treatment with etoposide for 72 hours in a range of concentrations 0.02-200 microM/L. Actin was visualised by fluorescence microscopy and streptavidingold method. These findings indicate that changes in actin after treatment with etoposide were dose-dependent. Significant changes in the cellular distribution of F-actin in K-562 cells were obtained after treatment with 20 and 200 microM/L etoposide. In comparison with control cells, the number of the cells decreased and cells were larger especially at 200 microM/L. F-actin was diffusely distributed throughout the cell at 20 microM/L. Treatment of cells with 200 microM/L etoposide showed F-actin diffusely distributed throughout the cell with local actin assemblies and also at the cell periphery. Immunogold labelling of actin was observed in cells treated with all doses of etoposide and control cells. Labelling was found in the nucleus and also in the cytoplasm. At the ultrastructural level, cells treated with 200 microM/L etoposide showed protrusions at the surface, in which increase of actin was often observed. Etoposide causes changes in actin distribution of K-562 cells, and the changes in expression of actin were not only restricted to cell with features of apoptosis.

[Apoptosis of K562 cells induced by antisense-oligonucleotides of CRKL gene].

BACKGROUND & OBJECTIVE: It was recently reported that the proteins coded by CRKL (CT10 regulator of kinase like, CRK like) gene play an important role in the pathogenesis of chronic myeloid leukemia (CML). This study was designed to investigate the effect of antisense oligonucleotides of CRKL gene (CRKL-ASDON) on CML K562 cell lines. METHODS: K562 cells were transfected with CRKL-ASDON, using liposome as the vector. The changes of cell morphology, cell cycle, and gene expression were observed through living cell count, transmission electron microscopy, flow cytometry (FCM), DNA Ladder assay. RESULTS: Under the action of CRKL-ASDON, growth of K562 cell was markedly inhibited. An apoptotic peak appeared before diploid peak in FCM; various apoptotic stages of K562 cells were observed under electron microscope; over ladder-shaped band of cell apoptosis was seen in abstracted DNA of the gene group by gel electrophoresis. Meanwhile, the above changes did not appear in the control. CONCLUSION: CRKL gene is an important factor in the pathogenesis of Ph-positive CML.

Direct current decreases cell viability but not P-glycoprotein expression and function in human multidrug resistant leukemic cells.

Inhibition of tumor growth induced by treatment with direct current (DC) has been reported in several systems. In the current work, the cellular effects generated by the DC treatment of the human leukemic K562 cell line and its vincristine-resistant derivative K562-Lucena 1 were analyzed by trypan blue staining and transmission electron microscopy. DC stimulation induced cell lysis, alterations in shape, membrane extraction or discontinuity, and intense vacuolization of some cells. In addition, treatment of K562 and K562-Lucena 1 cells caused a marked decrease in viability. Since multidrug resistance is a major factor contributing with failure of chemotherapy in many tumors, the expression and function of P-glycoprotein (P-gp) in K562-Lucena 1 cells were also studied. The expression of mdr1, the gene encoding P-gp, was analyzed by reverse transcription polymerase chain reaction, which showed that this gene was equally expressed in either treated or untreated cells. These results were confirmed by flow cytometry with a monoclonal anti P-gp antibody and the Rhodamine 123 extrusion method, which revealed that P-gp surface expression and function were unaltered after DC treatment. Our results suggest that DC treatment does not affect P-gp in human leukemic cells, but affects their viability by mechanisms that would involve clear cellular effects, but also additional targets, whose relevance in dc treated tumoral cells is currently discussed.

Complete karyotype characterization of the K562 cell line by combined application of G-banding, multiplex-fluorescence in situ hybridization, fluorescence in situ hybridization, and comparative genomic hybridization.

This study combines conventional cytogenetics, fluorescence in situ hybridization (FISH), multiplex-FISH and comparative genomic hybridization (CGH). In applying this multimodal approach on the human leukemia cell line K562, the chromosome composition was refined in detail and compared with data from the literature. A hypotriploid karyotype with a modal chromosome number of 67, and 21 unique marker chromosomes were identified. The classification of six markers was identical to published data and the composition of five further markers from the literature could be fully clarified for the first time. The composition of another five markers, which have been interpreted in divergent ways in different studies, were elucidated without doubt. Finally, five new markers of our study seem to have no equivalents in former studies, very likely due to limitations of conventional cytogenetics. The combinatory application of complementary techniques as shown in this study will be very useful to provide the basis of a refined genotype analysis on the chromosomal level.

Modifications of mitochondria in human tumor cells during anthracycline-induced apoptosis.

Adriamycin (ADR), one of the major antitumor agents used for the clinical treatment of a wide variety of human cancers and its glutathione(GSH)-conjugated adduct, ADRIGLU, induced apoptosis in K562 erythroleukemia and TVM-A12 clone 2 melanoma human cell lines. We have previously reported that ADR has nuclear localization and that ADRIGLU localizes exclusively in the cytoplasm. During ADR or ADRIGLU treatment, significant depletion of the cell energy state, demonstrated by a reduction in high-energy phosphates (ATP and GTP) and a decrease in energy charge potential (ECP), were recorded between 2 hours and 24 hours, by HPLC analysis. Transmission electron microscopy also revealed that between 2 hours and 24 hours of ADR or ADRIGLU treatment, mitochondria underwent evident morphological changes, from an initial "high amplitude swelling state" to a "shrinkage state" and finally, in early apoptotic cells, to an "abnormal shrinkage state", in which a marked accumulation of pycnotic mitochondria was also observed. Confocal microscopic analysis, using the potential-sensitive dye JC-1, showed that inhibition of cell energy metabolism was preceded by a rapid decrease in mitochondrial transmembrane potential (delta psi m). With the progression of exposure time, the early depolarization of the mitochondrial membrane was followed by a transient reversion to normal delta psi m until, in apoptotic cells, almost all mitochondrial subpopulations appeared to be hyperpolarized. Our results indicated that mitochondria are actively involved in anthracycline-induced programmed cell death, suggesting a novel mechanism that may be common to all forms of apoptosis.

The expression of proliferating cell nuclear antigen (PCNA) in leukemia cell lines HL-60 and K-562 at the light and electron microscope level.

PCNA antigen was localized at the light and electron microscopes level in two human leukemia cell lines HL-60 and K-562. PCNA expression was used to discriminate cycling from non-cycling cells. PCNA protein at the level of the light microscope was present in 70% of the cell in HL-60 cell line and in 65% of the cells in K-562 line. Streptavidin immunogold method was used for localization of PCNA expression at the ultrastructural level. Positive staining for this protein was seen as granular pattern in the nucleus and in the cytoplasm. In the nucleus the gold particles were seen to be associated with heterochromatin and euchromatin of the leukemia cells. In cytoplasm it was found on the endoplasmic reticulum and associated with ribosomes. Controls of the leukemia cells incubation with normal mouse serum showed no labelling at the light and electron microscope level.

Nucleus labeling or membrane labeling for studying the proliferation of drug treated cells?

Proliferation and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukemia (AML). This study compared cell cycle analysis (nuclear labeling) with cell division analysis (membrane labeling, PKH67) for studying the proliferation of cells cultured with daunorubicin (DNR) and/or Cytarabine (Ara-C), drugs commonly used in AML treatment. PKHs are a family of lipophilic, fluorescent membrane intercalating dyes. When labeled cells divide, the resulting daughter cells receive half the label, reducing fluorescence intensity to one-half that of the parent cells. DNR has the disadvantage of overlapping the spectrum of propidium iodide (PI), which is the most commonly used marker of membrane integrity. In this study, necrosis was evaluated using TOTO-3, a marker of nucleic acids emitting fluorescence above 645 nm and which incorporates cells with disrupted membranes. Comparison of cell cycle analysis with cell division for studying proliferation revealed that PKH67 was a marker of choice for analyzing the mitotic process in cells cultured with drugs.

A unique monoclonal antibody mNI-11 rapidly enhances spread formation in human umbilical vein endothelial cells.

We previously reported a novel monoclonal antibody (MAb), designated mNI-11, recognizing an adhesion-associated antigen distinct from any previously reported ones. In this article, this adhesion-associated antigen with a molecular weight of about 97 kDa was found to be strongly expressed on human umbilical vein endothelial cells (HUVECs) by fluorescence-activated cell sorter (FACS) analysis. Expression of this antigen on HUVECs was slightly increased in response to the exposure to tumor necrosis factor-alpha (TNF-alpha) or phorbol myristate acetate (PMA). As a biological function exerted by this antigen, it was of great interest that immobilized mNI-11 directly and rapidly enhanced the spread formation of HUVECs, whereas MAbs binding other adhesion-associated antigens such as mNI-58A (anti-CD11a), L130 (anti-CD18), L133.1 (anti-CD31), L178 (anti-CD44), L25.3 (anti-CD49d), or LB-2 (anti-CD54) did not carry such activity under the same conditions. The HUVECs spread formation enhanced by mNI-11 was completely blocked in the presence of a microfilament formation inhibitor, cytochalasin D (CyD), a Ca2+ calmodulin inhibitor, W-7, EDTA, and was partially blocked by a microtubule formation inhibitor, nocodazole, a protein kinase C (PKC) inhibitor, H-7, and a protein synthesis inhibitor, cycloheximide (CHX). However, a protein tyrosine kinase (PTK) inhibitor, genistein, did not affect the spread formation under the same conditions. Taken together, it was suggested that the spread formation of HUVECs enhanced by mNI-11 was mainly associated with the influx of Ca2+ and microfilament reorganization. In addition, the novel property associated with mNI-11 to enhance the spread formation of HUVECs was possibly mediated through its reaction against a unique epitope on HUVECs.

Fern spore extracts can damage DNA.

The carcinogenicity of the vegetative tissues of bracken fern (Pteridium) has long been established. More recently, the carcinogenic effects of the spores of bracken have also been recognized. Both vegetative tissues and spores of bracken can induce adducts in DNA in animal tissues, but the possible genotoxic or carcinogenic effects of spores from fern species other than bracken are unknown. The single-cell gel electrophoresis ('comet') assay was used to investigate whether fern spores can cause DNA damage in vitro. Extracts of spores from six fern species were administered to cultured human premyeloid leukaemia (K562) cells. Spore extracts of five fern species: Anemia phyllitidis, Dicksonia antarctica, Pteridium aquilinum, Pteris vittata and Sadleria pallida, induced significantly more DNA strand breaks than those in the control groups. Only in one species, Osmunda regalis, was the effect no different from that in the control groups. Using extracts from A. phyllitidis and P. vittata, the extent of DNA damage was increased by increasing the original dose 10 times, whereas an experiment in which exposure times were varied suggested that the highest levels of strand breaks appear after 2 h exposure. Simultaneous incubation with human S9 liver enzyme mix ablated the damaging effect of the extracts. Our data show that fern spore extracts can cause DNA damage in human cells in vitro. Considering the strong correlation between DNA damage and carcinogenic events, the observations made in this report may well have some implications for human health.

[The effects of jiexinkang on the apoptosis of leukemic cells].

UNLABELLED: The effects of Chinese medicine jiexinkang(JXK) on apoptosis of leukemic cells were studied by morphology approach and electrophoresis of DNA fragments. RESULTS: 1. The apoptotic cells and apoptotic bodies were found by electrical microscopy and the typical ladders of DNA fragments were detected by electrophoresis. 2. JXK induced apoptosis of leukemic cells(HL-60 and K562) in a certain range of concentration and at appropriate time. The time to K562 cell apoptosis was longer than that of HL-60 and its dosage was larger than that of HL-60. CONCLUSION: HL-60 and K562 leukemic cell apoptosis may be induced by JXK and there is correlation between dose and time. The study provides experimental evidences for the clinical treatment of leukemia.