Death Of K562 Cells Research Articles

Imatinib induces mitochondria-dependent apoptosis of the Bcr-Abl-positive K562 cell line and its differentiation toward the erythroid lineage.

Imatinib has emerged as the lead compound for clinical development against chronic myeloid leukemia. Imatinib inhibits the kinase activity of Bcr-Abl, which functions by enhancing the proliferation of hematopoietic precursors and protecting them against apoptosis. Imatinib induces apoptosis of Bcr-Abl positive cells, but how the drug effectively kills these cells remains partially understood. We show here that in K562 cells imatinib i) abolished Bcr-Abl phosphorylation and activity and as a consequence Erk1/2, JNK, and AKT activation; ii) induced mitochondrial transmembrane permeability dissipation; iii) activated caspases 3, 9, and 8, demonstrating that the effect of imatinib is integrated at the mitochondrial level; and iv) triggered caspase-dependent cleavage of Bcr-Abl. Interestingly, imatinib-mediated apoptosis was accompanied by erythroid differentiation of K562 cells. Moreover, phorbol esters inhibited imatinib-induced cell death and promoted differentiation toward the megakaryocytic lineage. Finally, we determined by c-DNA array analysis that more than 20 genes were modulated by imatinib. These genes are involved in both cell death and differentiation programs, and some of them have never been reported before to be expressed or involved in erythroid differentiation. Our results demonstrate that imatinib is responsible for a major modification of the genetic program resulting in death and/or differentiation of K562 cells.

Activation of natural killer cells with interleukin 2 (IL-2) and IL-12 increases perforin binding and subsequent lysis of tumour cells.

Natural killer (NK) cells can lyse a variety of different tumour cells by exocytosis of perforin, subsequent binding of perforin to the target cell membrane and formation of lytic pores. Some tumour cells, however, are resistant to cellular cytotoxicity. Using the NK-resistant tumour cell lines ML-2, MONOMAC-1, RPMI and L540Cy, we demonstrated that activation of NK cells with interleukin 2 (IL-2) and IL-12 resulted in significant lysis of these tumour targets. To investigate the underlying mechanisms, we isolated the cytotoxic granules from non-activated and IL-2-/IL-12-activated NK cells and compared the killing of K562 leukaemia cells (sensitive to NK cell-mediated lysis) and ML-2 leukaemia cells (resistant to NK cell-mediated lysis). In contrast to K562 cells, which were easily killed by NK-cell granules, ML-2 cells were resistant to granules from non-activated NK cells. However, granules from NK cells activated with IL-2 and IL-12 were able to induce significant tumour cell lysis. Cell death of both K562 and ML-2 cells by granules from activated NK cells was completely blocked by anti-perforin antibodies, indicating that perforin mainly accounts for the lysis induced by NK granules. Comparing granules from non-activated and IL-2-/IL-12-activated NK cells, the increased cell death of ML-2 cells was caused by an improved binding of perforin to the target cell membrane. Functional assays, however, indicated that the differences in perforin binding were not as a result of an augmented production of perforin by activated NK cells. We conclude that activation of NK cells results in an increased binding of perforin and subsequent lysis of tumour cells.

Glutathione depletion restores the susceptibility of cisplatin-resistant chronic myelogenous leukemia cell lines to Natural Killer cell-mediated cell death via necrosis rather than apoptosis

We investigated the effect of intracellular glutathione (GSH) levels on Natural Killer-mediated apoptosis in cisplatin-resistant K562 cells. K562/B6 and K562/C9 are cisplatin-resistant K562 cells less susceptible to lysis by natural killer cells. Cisplatin-resistant K562 cells did not present the apoptotic pattern of DNA fragmentation as it was observed for their maternal counterparts. K562/B6 and K562/C9 cell lines produce 1.6- and 1.9-times more GSH than K562 cells. Treatment of both cell lines with D,L-buthionine-(S,R)-sulfoximine (BSO, a gamma-glutamyl cysteine synthetase inhibitor) decreased GSH levels and augmented cell death induced by NK cells via a necrotic rather than an apoptotic process. Proliferating cell nuclear antigen (PCNA) expression was elevated in cisplatin-resistant K562 subclones, and the reduction of GSH levels after treatment with BSO decreased the expression of PCNA. These results suggest that the GSH level affects the NK cell-mediated cell death of cisplatin-resistant K562 cells by inducing necrosis rather than apoptosis.

Augmentation of methylprednisolone-induced differentiation of myeloid leukemia cells by serine/threonine protein phosphatase inhibitors

To elucidate the roles of serine/threonine protein phosphatases type 1 (PP1) and type 2A (PP2A) in methylprednisolone-induced differentiation of HL60 cells into granulocytes and K562 cells into monocytes, we examined the effect of serine/threonine protein phosphatase inhibitors, okadaic acid and Cal-A on the proliferation/ differentiation of HL60 and K562 cells. Okadaic acid and Cal-A augmented methylprednisolone induced granulocytic differentiation and cell death of HL60 cells and monocytic differentiation and cell death of K562 cells in different dose ranges, respectively. These data suggest an important role of PP1 and PP2A in the mechanism leading to differentiation of leukemic cells.

Nitric oxide as a bifunctional regulator of apoptosis.

It was inevitable that important relationships between two of the most intensely studied topics in biomedical research, apoptosis and nitric oxide (NO), would become apparent. Apoptosis is essential to normal development as well as physiological cell turnover. Although apoptosis in excess can manifest as tissue damage, a failure to undergo apoptosis constitutes pathological cellular overgrowth. It is now evident that NO and its reaction products can either promote or prevent apoptosis in a multitude of settings. The ubiquitous distribution of the NO synthases and the remarkable diffusibility and diverse chemical reactivity of NO in biological systems make this molecule unique among the regulators of apoptosis. Understanding the factors that govern the consequences of NO exposure on cell viability and identifying the conditions in which NO regulation of apoptosis contribute to pathology are topics of considerable interest and potential importance. In this article, we will review the recent observations on NO as a regulator of apoptosis. Apoptosis, or programmed cell death, is distinguished from lytic or necrotic cell death by specific biochemical and structural events (see recent review in Reference 11 ). Apoptogenic signals trigger cell-specific signaling pathways, including protease activation, followed by the appearance of morphological changes characteristic of cells undergoing apoptosis, including condensation of nuclei and cytoplasm, blebbing of the cytoplasmic membranes, and finally fragmentation into apoptotic bodies that are phagocytosed by neighboring cells. The elucidation of the signaling events in apoptosis is occurring at a rapid pace and includes the identification of the key roles of cysteine proteases (known as caspases), Bcl-2 family members, and mitochondria. Caspases, the mammalian counterpart of ced-3 in Caenorhabditis elegans , are a family of cysteine proteases now known to contain at least 14 homologs. Ectopic expression of any of the caspase family proteases can cause apoptosis; however, not all caspase family …

Activation of nitric oxide synthase is involved in tamoxifen-induced apoptosis of human erythroleukemia K562 cells

Tamoxifen induces apoptosis (programmed cell death) in human erythroleukemia K562 cells. Nitric oxide synthase activity and expression increased in apoptotic cells by 315% and 280%, respectively, compared to controls. The specific inhibitor of nitric oxide synthase, l-NAME, protected K562 cells from tamoxifen-induced apoptosis, whereas the nitric oxide donor, sodium nitroprusside (SNP), potentiated the apoptotic effect of the drug. In addition, 5-lipoxygenase was activated by tamoxifen and the specific enzyme inhibitor, MK886, protected K562 cells against the drug. Conversely, the 5-lipoxygenase product, 5-hydroperoxyeicosatetraenoic acid, enhanced the tamoxifen-induced apoptosis. Finally, tamoxifen altered also membrane properties of K562 cells.

Cholesterol, but not its esters, triggers programmed cell death in human erythroleukemia K562 cells.

Cholesterol, its biosynthetic precursors and the cholesterol-lowering drug compactin were able to inhibit the growth of human erythroleukemia K562 cells. Compactin, farnesyldiphosphate and cholesterol were cytotoxic by the induction of apoptosis (programmed cell death, PCD). Compactin doubled the number of apoptotic cells compared to control numbers, whereas farnesyldiphosphate and cholesterol led to a fivefold increase in PCD over the control levels. At variance with cholesterol, cholesterol esters did not affect K562 cell viability and apoptotic body formation, regardless of chain length and degree of saturation. Compactin and farnesyldiphosphate reduced the membrane cholesterol content, thus increasing membrane fluidity. Conversely, cholesterol treatment reduced the membrane fluidity by increasing cholesterol content in the lipid bilayer. Unlike farnesyldiphosphate, the other cholesterol precursors and cholesterol esters were ineffective in increasing the cholesterol content and, thereby, the fluidity of cell membranes. Compactin and cholesterol precursors, apart from farnesyldiphosphate, did not affect the amount of the farnesylated proteins Ras and lamin B in the cytosolic and the membrane fractions of K562 cell extracts, whereas farnesyldiphosphate reduced the content of both proteins in both fractions. The level of lamin B in K562 cytosol and membranes was also reduced by cholesterol treatment, which did not significantly affect the amount of Ras. These findings highlight the role of cholesterol in promoting PCD.

Apoptosis of human myeloid leukemia cells induced by an inhibitor of protein phosphatases (okadaic acid) is prevented by Bcl-2 and Bcl-X(L).

Okadaic acid, an inhibitor of serine/threonine protein phosphatases 1 and 2A has been shown to cause mitotic arrest and cell death of HL-60 and K562 cells. HL-60 cells express Bcl-2 and little or no Bcl-X(L), while K562 expresses Bcl-X(L) but not Bcl-2. Since phosphorylation/dephosphorylation reactions have been suggested to be involved in the regulation of Bcl-2, we planned to investigate whether the expression of Bcl-2, Bcl-X(L) and Bax, a protein that antagonizes the antiapoptotic function of Bcl-2, are regulated in myeloid leukemia cell lines (K562, KU812 and HL-60) treated with okadaic acid. Our results indicate that exposure of all three leukemic cell lines to nanomolar concentrations of okadaic acid causes a loss of viability by activation of an apoptotic process accompanied by a marked decrease in the expression of Bcl-2, Bcl-X(L) and Bax at both mRNA and protein level, but not of c-fos, vimentin and epsilon-globin, ruling out a non-specific effect of okadaic acid. Furthermore, constitutive expression of either Bcl-X(L) or Bcl-2 by gene transfer inhibited apoptosis triggered by okadaic acid in K562 cells. Thus, we suggest that protein phosphatases may be involved in maintaining the expression of bcl-2 family genes as part of the survival machinery of the cell.

BCR-ABL Does Not Prevent Apoptotic Death Induced by Human Natural Killer or Lymphokine-Activated Killer Cells

The erythromyeloid cell line, K562, the most sensitive target in human natural killer (NK) cell mediated cytotoxicity, is derived from a chronic myeloid leukemia (CML) patient and expresses the characteristic reciprocal translocation t(9;22). The resulting BCR-ABL fusion protein has been shown to mediate the unusual resistance of K562, and other BCR-ABL expressing lines, to apoptosis induced by a variety of agents (irradiation, UV light, cytotoxic drugs). Here we show that human NK and lymphokine-activated killer (LAK) cells, when tested at low effector to target ratio, can readily induce apoptotic death in K562 cells. This was accompanied with classical DNA oligonucleosomal fragmentation, an unexpected finding given the reported lack of such fragmentation when apoptosis is induced in K562 by chemical agents, after downregulation of BCR-ABL. Apoptosis was assessed by several means: morphological studies, 125I-DNA versus 51Cr release, DNA agarose gel electrophoresis, and results were always concordant, with a delayed kinetics for DNA oligonucleosomal fragmentation. Similar data were obtained with a pluripotent human hematopoietic cell line, UT-7, infected with a defective amphotropic p210 BCR-ABL retrovirus. The BCR-ABL expressing subclone UT-7/9, while being no longer sensitive to cytotoxic drugs or to tumor necrosis factor, a lytic mediator to which UT-7 cells are sensitive, underwent apoptotic death when exposed to LAK effector cells to the same degree as the parental UT-7 line. With these targets, DNA oligonucleosomal fragmentation occurred concomitantly with isotope release. Results obtained with several inhibitors of exocytosis strongly suggest that cytotoxic granules mediate NK and LAK cell-induced apoptotic death. In conclusion, NK and LAK cell-induced apoptotic signals, unlike those activated by chemotherapeutic agents, are unaffected by the antiapoptotic action of BCR-ABL. This unique property may support the observed curative effect of allogeneic bone marrow transplantation in CML.

Stimulation of Interleukin-1β-Converting Enzyme Activity during Growth Inhibition by CPT-11 in the Human Myeloid Leukemia Cell Line K562

Camptothecin (CPT) was first extracted fromCamptotheca acuminataand has a strong antitumor effect. Its water-soluble derivative, CPT-11, has higher therapeutic efficacy and less toxicity than CPT. Recently, CPT-treated cells have been shown to undergo apoptosis. However, the mechanism of induction of apoptosis by CPT has not been characterized in detail in any type of cells. On the other hand, interleukin-1β-converting enzyme (ICE) is a mammalian homologue of CED3, a protein required for apoptosis in the nematodeCaenorhabditis elegans.To determine how CPT-11 brings about cell death by apoptosis, we investigated the effects of CPT-11 on the expression of ICE activity in K562 cells, which represent human myeloid leukemia cells. The proliferation of K562 cells was shown to be inhibited by the presence of CPT-11 in the culture medium. We also found that the levels of mRNA for ICE in the cells were increased in the presence of CPT-11. Furthermore, we demonstrated that when CPT-11 was added to the culture medium, apoptosis of K562 cells was clearly detectedin situ.These features suggested that CPT-11 enhances the apoptotic cell death in K562 cells and that a part of induction of apoptosis by CPT-11 may be correlated with the stimulation of the ICE activity.