Novel immunologic tolerance of human cancer cell xenotransplants in zebrafish.

Immune deficiency or suppression in host animals is an essential precondition for the success of cancer cell xenotransplantation because the host immune system has a tendency to reject implanted cells. However, in such animals, the typical tumor microenvironment seen in cancer subjects does not form because of the lack of normal immunity. Here, we developed a novel zebrafish (Danio rerio) model based on 2 rounds of cancer cell xenotransplantation that achieved cancer-specific immunologic tolerance without immunosuppression. We irradiated human cancer cells (PC-3, K562 and HepG2) to abolish their proliferative abilities and implanted them into zebrafish larvae. These cells survived for 2 weeks in the developing host. Three months after the first implantation, the zebrafish were implanted with the same, but nonirradiated, cell lines. These cancer cells proliferated and exhibited metastasis without immune suppression. To reveal the transcriptional mechanism of this immune tolerance, we conducted dual RNA-seq of the tumor with its surrounding tissues and identified several regulatory zebrafish genes that are involved in immunity; the expression of plasminogen activator, urokinase, and forkhead box P3 was altered in response to immunologic tolerance. In conclusion, this xenograft method has potential as a platform for zebrafish-based anticancer drug discovery because it can closely mimic human clinical cancers without inducing immune suppression.

Evaluation of the spatial distribution of gammaH2AX following ionizing radiation.

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR). The phosphorylated form of H2AX, referred to as gammaH2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorescence microscopy. Analysis and quantitation of gammaH2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds. Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of gammaH2AX as molecular marker of DSBs, a disparity in gamma-irradiation-induced gammaH2AX foci formation in euchromatin and heterochromatin has been observed. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of gammaH2AX following ionizing radiation. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between gammaH2AX formation and active transcription. Here we demonstrate our immunofluorescence method for detection and quantitation of gammaH2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Bystander effects induced by medium from irradiated cells: similar transcriptome responses in irradiated and bystander K562 cells.

PURPOSE: Cells exposed to ionizing radiation release factors that induce deoxyribonucleic acid damage, chromosomal instability, apoptosis, and changes in the proliferation rate of neighboring unexposed cells, phenomena known as bystander effects. This work analyzes and compares changes in global transcript levels induced by direct irradiation and by bystander effects in K562 (human erythroleukemia) cells. METHODS AND MATERIALS: Cells were X-irradiated with 4 Gy or transferred into culture medium collected from cells 1 h after irradiation (irradiation-conditioned medium). Global transcript profiles were assessed after 36 h of growth by use of Affymetrix microarrays (Affymetrix, Santa Clara, CA) and the kinetics of change of selected transcripts by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: The level of the majority (72%) of transcripts changed similarly (increase, decrease, or no change) in cells grown in irradiation-conditioned medium or irradiated, whereas only 0.6% showed an opposite response. Transcript level changes in bystander and irradiated cells were significantly different from those in untreated cells grown for the same amount of time and were confirmed by quantitative reverse transcriptase-polymerase chain reaction for selected genes. Signaling pathways in which the highest number of transcripts changed in both conditions were found in the following groups: neuroactive ligand-receptor, cytokine-cytokine receptor interaction, Janus Kinase-Signal Transducers and Activators of Transcription (JAK-STAT) and Mitogen-Activated Protein Kinase (MAPK) In control cells more transcripts were downregulated than in irradiated and bystander cells with transcription factors YBX1 and STAT5B, heat shock protein HSPA1A, and ribonucleic acid helicase DDX3X as examples. CONCLUSIONS: The transcriptomes of cells grown in medium from X-irradiated cells or directly irradiated show very similar changes. Signals released by irradiated cells may cause changes in the transcriptome of neighboring cells that sustain their survival.

5-Aminolaevulinic acid enhances ultrasound-induced mitochondrial damage in K562 cells.

BACKGROUND: Ultrasound therapy is a new modality in the control of malignant cancers. The aim of the present study was to investigate the effect of 5-aminolaevulinic acid on the ultrasonic killing action in the cancer cells. MATERIALS/METHODS: The K562 cells as a cancer cell model were subjected to investigate the effect of 5-aminolaevulinic acid (5-ALA) on the ultrasonic killing action, in which the 5-ALA concentration was 2mM and the ultrasound exposure was 15 s at the intensity of 0.46 W/cm(2) and the frequency of 1.7MHz. Cytotoxicity was investigated 24h after ultrasound exposure using the trypan blue exclusion test. Ultrastructural cell morphology and mitochondrial changes were observed using transmission electron microscopy (TEM). Mitochondrial membrane potential (DeltaPsim) was evaluated using Rhodamine 123 assay. RESULTS: The death rates of the K562 cells in the controls including sham radiation and 5-ALA treatment alone were 1.81+/-0.13%, 1.27+/-0.20%, respectively. Those in ultrasound radiation alone and 5-ALA-ultrasound treatment were 12.61+/-2.63%, 46.87+/-4.09%, respectively. There were significant differences between 5-ALA-ultrasound treatment, ultrasound radiation alone and the controls (P<0.05). TEM showed that the mitochondria expanding and some vacuoles were found in the ultrasound-treated cells. After the treatment of ultrasound and 5-ALA together some cells presented typical characteristics of apoptotic cells, such as nuclear condensation and crescent formation. Mitochondria of the cells were damaged more seriously than those treated by ultrasound alone, there were obvious swollen mitochondria and mitochondria in which cristae were almost perfectly disappeared, and more vacuolar mitochondria were founded. Mitochondrial membrane potential (DeltaPsim) was more significantly collapsed when the K562 cells were exposed to 2mM 5-ALA for 4h and then 0.46 W/cm(2) irradiation of ultrasound than ultrasound radiation alone. CONCLUSION: 5-ALA pretreatment significantly enhanced the cytotoxicity of ultrasound radiation in K562 cells. The damage of mitochondria structure and function might be an important cause of cell death in K562 cells induced by the treatment of ultrasound radiation and 5-ALA together.

[Effects of PUVA on expression of Fas and FasL in inducing apoptosis of NB4 and K562 cells].

OBJECTIVE: To explore the effects of psoralen (PSO) and long wave ultraviolet A (PUVA) on expression of Fas and FasL in apoptosis of NB4 and K562 leukemia cells. METHODS: The NB4 and K562 cells were taken as the study objects and their apoptosis ratios, ultrastructure changes and the expression of Fas and FasL were detected in order to observe the effects of PSO and UVA of wave length 360 nm on human leukemia cells. The factorial design and analysis of variance were used to analyze the interaction among the factors. RESULTS: All of PSO, UVA and PUVA could induce the apoptosis of NB4 and K562 cells, and the effects of PUVA were stronger than the other two. After treated with PUVA, the NB4 and K562 cells all showed obvious ultrastruture changes about apoptsis under the electron microscope. All of PSO, UVA and PUVA could increase the expression of Fas gene and protein, and decrease the expression of FasL gene and protein. Moreover, the effects of PUVA were stronger than the other two. CONCLUSION: PUVA can induce the apoptosis of NB4 and K562 cells and the effects are the strongest, one of the pathway of PUVA to induce apoptosis is to upregulate the expression of Fas gene and downregulate the expression of FasL gene.

Nuclear organization of PML bodies in leukaemic and multiple myeloma cells.

The nuclear arrangement of promyelocytic leukaemia nuclear bodies (PML NBs) was studied in vitro after the cell treatment by clinically used agents such as all-trans retinoic acid (RA) in human leukaemia and cytostatics or gamma radiation in multiple myeloma cells. In addition, the influence of phorbol ester (PMA) on PML NBs formation was analyzed. A reduced number of PML bodies, which led to relocation of PML NBs closer to the nuclear interior, mostly accompanied RA- and PMA-induced differentiation. Centrally located PML NBs were associated with transcriptional protein RNAP II and SC35 regions, which support importance of PML NBs in RNA processing that mostly proceeds within the nuclear interior. Conversely, the quantity of PML NBs was increased after cytostatic treatment, which caused re-distribution of PML NBs closer to the nuclear envelope. Here we showed correlations between the number of PML NBs and average Centre-to-PML distances. Moreover, a number of cells in S phase, especially during differentiation, influenced number of PML NBs. Studying the proteins involved in PML compartment, such as c-MYC, cell-type specific association of c-MYC and the PML NBs was observed in selected leukaemic cells undergoing differentiation, which was accompanied by c-MYC down-regulation.

Differential lysis of tumors by polyclonal T cell lines and T cell clones specific for hTERT.

The human telomerase reverse transcriptase hTERT is overexpressed in most human tumors and contributes importantly to oncogenesis by maintaining the integrity of telomeric DNA. Despite being a self-antigen, the hTERT enzyme is immunogenic. Peptides derived from hTERT have been shown both in vitro and in vivo to drive the activation and proliferation of peptide-specific T lymphocytes. An HLA-A2-binding peptide from Htert (I540, ILAKFLHWL) has been used to generate peptide-specific T cells in vitro and in vivo in patients that lyse telomerase-positive tumors in an MHC-restricted fashion. Although these and other data suggest that I540 is naturally processed and presented on the surface of certain tumor cells, there are reports that I540-specific T cells, and in particular, T cell clones, do not lyse tumors in vitro. Here, we compared cytotoxic function of I540-specfic T cell clones vs. polyclonal T cell lines, including clones and lines generated from the same donor. We found that I540-specific polyclonal T cell lines lyse telomerase-positive tumors whereas non-specific polyclonal T cell lines and I540-specific T cell clones do not. Estimated TCR avidity for I540, as well as cell surface expression of CD45RO, CD45RA, CD28, CD27, CD57 and CD62L were similar between lines and clones. V beta usage, however, differed such that the majority of the I540-specific TCR repertoire found in polyclonal T cell lines was not represented in clones generated from the same source material. Thus, I540-specific T cells can vary in cytotoxic potential depending on the method of generation, isolation and expansion.

Photodynamic treatment (ALA-PDT) suppresses the expression of the oncogenic Bcr-Abl kinase and affects the cytoskeleton organization in K562 cells.

K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation. It was followed by a necrosis-like cell death [K. Kuzelová, D. Grebenová, M. Pluskalová, I. Marinov, Z. Hrkal, J. Photochem. Photobiol. B 73 (2004) 67-78]. ALA-PDT led to the perturbation of the Hsp90/p23 multichaperone complex of which the Bcr-Abl is the client protein. Bcr-Abl protein was suppressed whereas the bcr-abl mRNA level was not affected. Further on, we observed several changes in the cytoskeleton organization. We detected ALA-PDT-mediated disruption of filamental actin structure using FITC-Phalloidin staining. In connection with this we uncovered certain cytoskeleton organizing proteins involved in the cell response to the treatment. Among these proteins, Septin2, which plays a role in maintaining actin bundles, was suppressed. Another one, PDZ-LIM domain protein 1 (CLP36) was altered. This protein acts as an adaptor molecule for LIM-kinase which phosphorylates and thus inactivates cofilin. Cofilin was indeed dephosphorylated and could thus be activated and operate as an actin-depolymerizing factor. We propose the scheme of molecular response of K562 cells to ALA-PDT.

DNA targeted UVA photosensitization: characterization of an extremely photopotent iodinated minor groove binding DNA ligand.

Previous studies have described UVA-induced DNA strand breakage at the binding sites of iodinated DNA minor groove binding bisbenzimidazoles. The DNA breakage, presumably mediated by the carbon-centred ligand radical produced by photodehalogenation, was also shown to be cytotoxic. The earlier studies included a comparison of three ligand isomers, designated ortho-, meta- and para-iodoHoechst, and the efficiency of photo-induction of strand breaks in plasmid DNA proved to be much higher for the ortho-isomer. We have now extended the comparison of the three isomers with respect to photo-induced cytotoxicity in K562 cells. Although the relationship between the extent of nuclear uptake and the concentration of the ligand in the medium was similar for the three isomers, assay of in situ dehalogenation in drug-treated cells indicated that the apparent cross-section for dehalogenation of the ortho-isomer was greater than 5-fold higher than that for the meta- and para-isomers. Also, analysis of clonogenic survival data showed that the dehalogenation event associated with ortho-iodoHoechst was a more efficient mediator of UVA-induced cytotoxicity in K562 cells than that for meta- or para-iodoHoechst. The number of dehalogenation events associated with 50% cell-kill for ortho-iodoHoechst (1.23+/-0.04 x 10(4)) was less than that for the para- (3.92+/-0.29 x 10(4)) and meta- (11.6+/-0.90 x 10(4)) isomers. Thus it is concluded that the photopotency of ortho-iodoHoechst, which is an important feature in the context of its potential use in clinical phototherapy, is due not only to more efficient UVA-mediated dehalogenation of the ligand, but also to greater cytotoxic potency per dehalogenation event.

Method of laser activated nano-thermolysis for elimination of tumor cells.

We describe novel ex vivo method for elimination of tumor cells from cell suspension, Laser Activated Nanothermolysis and propose this method for purging of bone marrow and blood transplants. K562 and human lympholeukemia cells were eliminated in experiments by laser-induced micro-bubbles that emerge inside individual target cells around selectively formed clusters of light-absorbing gold nanoparticles. Pretreatment of tumor cells with specific monoclonal antibodies and Ig-conjugated 30-nm gold particles allowed the formation of clusters of 10-20 on the surface of cell membrane. Electron microscopy found the nanoparticulate clusters inside the cells. Total (100%) elimination of K562 cells targeted with specific antibodies was achieved with single laser pulses with optical fluence of 5J/cm(2) at the wavelength of 532 nm without damage to the same cells targeted without specific antibodies. Total elimination of human lymphoblasts from suspension of normal stem cells was achieved by a single laser pulse with the optical fluence of 1.7J/cm(2), while the damage level of normal cells was 16%.

[Growth-inhibiting effect of psoralen plus ultraviolet-A light therapy on K562 cells].

OBJECTIVE: To observe the effects of psoralen plus ultraviolet-A light (PUVA) on K562 cells and the relative mechanism. METHODS: The effects of psoralen, ultraviolet-A light and PUVA on K562 cells were assayed by monotetrazolium test (MTT). DNA content was analyzed by flow cytometry (FCM). The apoptotic rates of K562 cells treated with 40 and 80 microg/ml psoralen for 24 and 48 hours were assayed by Annexin-V-FITC/PI reagent kit on FCM respectively. The ultrastructures of apoptotic cells were observed by a transmission electron microscope (TEM). RESULTS: Either single psoralen therapy or single ultraviolet-A irradiation had inhibiting effect on K562 cells. The inhibiting effect of PUVA on K562 cells was stronger than that of the single psoralen therapy or single ultraviolet-A light irradiation (P<0.05). Apoptotic peak (AP) was detected by FCM. TEM test showed that K562 cells treated with PUVA were smaller, having condensed cell nucleus, assembled chromatin, disintegrated nucleus body and the majority of the cells appeared to be apoptotic conformation. CONCLUSION: Psoralen has inhibiting effect on K562 cells, and the effect of PUVA is more significant. It is suggested that 10 min irradiation and 40 microg/ml terminal concentration of psoralen be probably the best choice for PUVA. The inhibiting effect of PUVA is due to apoptosis.

The histone deacetylase inhibitor, Trichostatin A, enhances radiation sensitivity and accumulation of gammaH2A.X.

Histone deacetylase inhibitors have been shown to induce numerous biologic effects including, altering cell cycle distribution, cytostasis and in certain cases apoptosis. Given their ability to disrupt critical biological processes in cancer cells, these agents are emerging as potential therapeutics for cancer. Recently, it has been identified that histone deacetylase inhibitors can also efficiently enhance the radiation sensitivity of cells, both in vitro and in vivo. In this study, we investigated whether the potent histone deacetylase inhibitor, Trichostatin A, modulates the radiation sensitivity of human erythroleukemic K562 cells. The endpoints we used were clonogenic survival, apoptosis and gammaH2AX immunoprecipitations of soluble chromatin. The findings from clonogenic survival assays indicated that incubation with Trichostatin A 24 hours prior to irradiation enhances the radiation sensitivity of K562 cells. The dose modification factors ranged from 1.1 when cells were incubated with 0.1 microM Trichostatin A to 2.3 at 1 microM Trichostatin A. Similarly, caspase-3 and caspase-7 assays indicated that Trichostatin A potentiates radiation-induced apoptosis in K562 cells, in a concentration dependent manner. Our results suggest the modulation of radiation effects observed at the lower Trichostatin A concentrations was associated with histone hyperacetylation and changes in phosphorylated gammaH2A.X formation on euchromatin. In contrast, at the higher Trichostatin A concentrations mechanisms such as drug-mediated cytotoxicity and G1 cell cycle arrest, contributed to the sensitization effect. More generally, our findings are consistent with those from recent studies and support the development of histone deacetylase inhibitors for use as radiation sensitizers, particularly for targeting radioresistant cancers.

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.

The effect of laser activation of 5,10,15,20-tetra-sulphophenyl-porphyrin loaded in K562 cells and human normal mononuclear cells.

Photodynamic therapy (PDT) is a relatively new type of treatment in cancer, based on a photosensitizer, visible light and molecular oxygen. Reactive oxygen species are generated, causing tumor cells death by apoptosis or necrosis. Significant nowadays research efforts are focused on finding new photosensitizers with antineoplastic activity and an acceptable toxicological profile. Although consistent information exists regarding PDT in solid tumors, relatively few data are available for PDT of blood cancers. Therefore, we carried out a comparative study on lymphoblastic K562 cells and human normal peripheral blood mononuclear cells (PBMC) treated at a density of 2 x 10(5) cells/mL with 5,10,15,20-tetra-sulphophenyl-porphyrin (TSPP) and then irradiated with He-Ne laser light (lamda = 632.8 nm). The following cell functions were investigated: viability, multiplication, RNA synthesis, total RNA levels and apoptosis. After irradiation, the viability of TSPP-loaded tumor cells decrease, the multiplication rate and the total RNA level are drastically reduced and cells undergo apoptosis. TSPP alone loaded into cells but not activated by irradiation, does not affect these cell parameters. Human normal PBMC subjected to TSPP loading and laser-irradiation develop a different cellular response, their viability and proliferative capacity not being altered by experimental PDT. Accordingly, it appears that TSPP is a non-aggressive compound for cellular physiology and becomes cytotoxic only by irradiation; moreover laser-activated TSPP affects only cells that have a tumoral pattern.

Levitation and movement of human tumor cells using a printed circuit board device based on software-controlled dielectrophoresis.

In this study we describe an original, efficient, and innovative printed circuit board (PCB) device able to generate dielectrophoresis-based, software-controlled cages that can be moved to any place inside a microchamber. Depending on their dielectrophoretic properties, eukaryotic cells can be "entrapped" in cages and moved under software control. The main conclusion gathered from the experimental data reported is that the PCB device based on dielectrophoresis permits levitation and movement of different tumor cells at different dielectrophoresis conditions. The results presented herein are therefore the basis for experiments aimed at forced interactions or separation of eukaryotic cells using "lab-on-a-chip." In fact, because many cages can be controlled at the same time, and two or more cages can be forced to share the same or a different location, it is possible, in principle, either to bring in contact cells of a differing histotype or to separate them.

ELF fields and photooxidation yielding lethal effects on cancer cells.

The lethal effect on human cancer cells was studied under three types of treatment: A) an ELF pulsed sinusoidal of 50 Hz electromagnetic field (PEMF) with amplitudes between 10 and 55 mT; B) the field and a cytostatic agent (actinomycin-C); and C) the field, the cytostatic agent, which has a photodynamic effect, and exposure to a halogen lamp. The results show a decreasing vitality of human K-562 and U-937 cancer cells in suspension with each additional treatment. Combination with other parameters as hyperthermia and/or hyperacidity could yield high killing rates by this noninvasive method.

Selective inhibition of tumoral cells growth by low power millimeter waves.

The effects of low power millimetric wave (MMW) radiation on the growth of tumor and healthy cells were studied. A wide-band frequency range between 53.57-78.33 GHz with a radiation density power of 27 x 10(-17) watt/Hz were used. The radiating energy was low enough not to increase the temperature of the cellular samples (cold irradiation). One hour of radiation treatment given every other day to three tumoral human stable cell lines, produced a noticeable inhibition of the cellular growth. The analogous treatment given to two healthy cell lines gave a weak growth stimulation. A scanning electron microscopy study of MCF-7-and K562-irradiated cells revealed that MMW irradiation induced profound morphological changes of the membrane. Finally, we also provided a mechanistic indication, based on millimeter wave spectroscopy of the cells: water is the primary absorber of these electromagnetic waves. Our work provides interesting evidence that wide band low power MMW irradiation, in the appropriate frequency range, could be used in the future as a cold means to cause selective inhibition of tumor cell growth.

Telomerase overexpression in K562 leukemia cells protects against apoptosis by serum deprivation and double-stranded DNA break inducing agents, but not against DNA synthesis inhibitors.

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusions. The maintenance of telomeres is essential for chromosomal stability. Telomerase is regulated by human telomerase reverse transcriptase (hTERT). c-Myc oncoprotein is also implicated in the positive regulation of hTERT expression. We show here that two clones of hTERT-transfected K562 erythroleukemia cells have elongated telomeres (22.5 and 24.0 kb), whereas telomere length of both c-Myc-transfected K562 cells and parental K562 cells is 6.5 kb. Telomerase activity and hTERT mRNA expression increased in hTERT-transfected K562 cells, while the expression levels of telomerase activity and hTERT in c-Myc-transfected K562 cells were similar to that in parental K562 cells, despite an overexpression of c-Myc. Importantly, we found that hTERT-transfected K562 cells are protected against apoptosis induced by serum deprivation and double-stranded DNA break inducing agents (ionizing irradiation, and etoposide (VP-16)), but not against DNA synthesis inhibitors (1-beta-D-arabinofuranosylcytosine and hydroxyurea). These findings suggest that overexpression of telomerase by transfecting hTERT confers telomere-elongation and resistance to double-stranded DNA break inducing agents.

DNA fragmentation induced in K562 cells by nitrogen ions.

This study was aimed at investigating the radiation induced DNA fragmentation pattern as a function of cellular differentiation and radiation quality. DNA double strand breaks (DSB) induced by gamma-rays were analyzed in K562 human proerythroblasts before (AP cells) and after (D cells) differentiation induction while DNA DSB induced by 125 keV/micrometers N-ions have been studied in AP cells. Pulsed-Field Gel Electrophoresis (PFGE) of cellular DNA was used to determine the DSB yield by analysis of the Fraction of Activity Released (FAR) and of the fragmentation pattern in a specific size range (5.7-0.225 Mbp). The results so far obtained show that the DSB induction by gamma-rays is different if evaluated with the FAR or with the fragmentation analysis. The DSB yield obtained with the former method is about 1.4 times higher in AP respect to D cells while the latter method indicates that more fragments are produced in D cells. Comparison between gamma-rays and N-ions in AP cells shows that no significant differences are detected by the FAR analysis; otherwise fragmentation analysis demonstrates a higher effectiveness of nitrogen ions.

[The MDR gene and cellular sensitivity to various effects]. / Gen mdr i chuvstvitel'nost' kletok k razlichnym vozdeistviiam.

mdr-Transfected K-562 cells revealed a relatively high resistance to cytotoxic monokines and ionizing radiation as compared to parental cells. Taken together with what is known about the resistance of mdr-expressing cells to multiple cytotoxic drugs, our results point to malignant cells having universal mechanisms of chemo-, bio- and radioresistance.