Characterization of imatinib-resistant K562 cell line displaying resistance mechanisms.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the t(9; 22) and the related oncogene, BCR-ABL. Tyrosine kinase activity of fusion protein BCR-ABL is the main cause of CML. Even if imatinib is used as a tyrosine kinase inhibitor (TKI) for CML therapy, drug resistance may occur in patients and the clinical failure of imatinib treatment in resistant patients had resulted with the use of another alternative TKIs. BCR-ABL dependent and independent molecular mechanisms have crucial roles in drug resistance. To reveal the underlying molecular mechanisms which play significant roles in imatinib resistance in CML, we established K562 imatinib-resistant cell line (K562r5) which was continuously exposed to (5µM) imatinib to investigate molecular mechanisms which play significant roles in drug resistance. First of all, we analyzed T315I, M351T, F315L and F359C/L/V mutations with DNA sequencing as a BCR-ABL dependent mechanism in our cell lines. Moreover, we investigated BCR-ABL independent mechanisms such as apoptosis, autophagy, drug transport and DNA repair which affect drug resistance in these cell lines. In vitro cell viability was determined by MTT assay. DNA sequencing analysis was performed to detect BCR-ABL mutations. The apoptotic effect of imatinib on CML cell lines was tested by flow cytometric Annexin V-PE staining and caspase activation assays. Apoptotic, autophagic, drug transporter and DNA repair genes expression levels were determined by RT-PCR. The conventional cytogenetic analysis was performed on K562s and K562r cells. Our results indicate that inhibition of apoptosis, induction of autophagy, overexpression of efflux gene MDR1 and down-regulation of influx gene OCT1 play crucial roles in the progression of imatinib resistance.

JAK2-V617F-mediated signalling is dependent on lipid rafts and statins inhibit JAK2-V617F-dependent cell growth.

Aberrant JAK2 signalling plays an important role in the aetiology of myeloproliferative neoplasms (MPNs). JAK2 inhibitors, however, do not readily eliminate neoplastic MPN cells and thus do not induce patient remission. Further understanding JAK2 signalling in MPNs may uncover novel avenues for therapeutic intervention. Recent work has suggested a potential role for cellular cholesterol in the activation of JAK2 by the erythropoietin receptor and in the development of an MPN-like disorder in mice. Our study demonstrates for the first time that the MPN-associated JAK2-V617F kinase localizes to lipid rafts and that JAK2-V617F-dependent signalling is inhibited by lipid raft disrupting agents, which target membrane cholesterol, a critical component of rafts. We also show for the first time that statins, 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to treat hypercholesterolaemia, induce apoptosis and inhibit JAK2-V617F-dependent cell growth. These cells are more sensitive to statin treatment than non-JAK2-V617F-dependent cells. Importantly, statin treatment inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but had no effect on erythroid colony formation from healthy individuals. Our study is the first to demonstrate that JAK2-V617F signalling is dependent on lipid rafts and that statins may be effective in a potential therapeutic approach for MPNs.

Ionizing radiation up-regulates telomerase activity in cancer cell lines by post-translational mechanism via ras/phosphatidylinositol 3-kinase/Akt pathway.

PURPOSE: Telomerase is considered currently as a hallmark of cancer, and its inhibition is expected to become an important anticancer modality. In contrast to abundant data concerning the effect of cytotoxic drugs on telomerase activity (TA), there is scant information on the effect of radiation on telomerase. The mechanism of telomerase regulation by irradiation has never been evaluated in detail. In the present study, we investigated the effect of radiation on TA and its regulation in cancer cells. EXPERIMENTAL DESIGN: The effect of various radiation doses on TA in several malignant and nonmalignant cell lines was evaluated. All malignant cells exhibited similar telomerase response to radiation and its regulation was assessed at transcriptional and post-translational levels in K562 cells. Next step was the evaluation of the upstream signaling pathways leading to changes in TA using kinetics and specific inhibitors. RESULTS: Radiation up-regulated TA in dose-dependent manner only in cancer cells. Telomerase was activated by phosphorylation by Akt and by cytoplasmic-nuclear shift. Transcriptional processes were not involved in TA. This telomerase regulation is mediated by Ras/phosphatidylinositol 3-kinase/Akt pathway. The canonical membrane effectors of irradiation (epidermal growth factor receptor, insulin-like growth factor-I receptor, and Ca2+ influx) were not involved in this process. CONCLUSIONS: Radiation up-regulates telomerase activity specifically in cancer cells. This study adds to accumulating evidence pointing to post-translational level as important mode of telomerase regulation. Telomerase activation due to radiation may be detrimental in treatment of cancer. Data described in this study may add to future interventions aiming at inhibition of telomerase activation during irradiation.

Antimony-trioxide- and arsenic-trioxide-induced apoptosis in myelogenic and lymphatic cell lines, recruitment of caspases, and loss of mitochondrial membrane potential are enhanced by modulators of the cellular glutathione redox system.

During the last years remission rates of more than 72% for arsenic(III)-oxide (As(2)O(3)) treatment in relapsed or refractory acute promyelocytic leukemia have been published. As(2)O(3) is under clinical investigation for therapy of leukemia and solid tumors. Due to the chemical affinity of arsenic and antimony, we analyzed the potency of antimony(III)-oxide (Sb(2)O(3)) to exert As(2)O(3)-like effects. Based on the same molar concentrations, lower efficacy in apoptosis induction and caspase-independent decrease of mitochondrial membrane potential was observed for Sb(2)O(3). No difference in sensitivity to As(2)O(3) or Sb(2)O(3) was detected in CEM cells when compared to their multiple drug resistant derivatives. Apoptosis was induced by combining sub-apoptotic concentrations of Sb(2)O(3) or As(2)O(3) with sub-apoptotic concentrations of DL: -buthionine-[S,R]-sulfoximine (BSO). Other modulators of the cellular redox system showed this effect to a lower extent and enhancement was not consistent for the different cell lines tested. Caspase inhibitors protected cell lines from Sb(2)O(3)- and As(2)O(3)-induced apoptosis. When BSO was added, the inhibitors lost their protective ability. The ability of modulators of the cellular redox system in clinically applicable concentrations to enhance the apoptotic effects of the two oxides in a synergistic way may be helpful to reduce their toxicity by optimizing their dose.

Cyclosporin A potentiates the cytotoxic effects of methyl methanesulphonate in HL-60 and K562 cells.

Methyl methanesulphonate (MMS) is a DNA damaging agent, which induces oxidative stress, ATP depletion, and consequently, cell death, in HL-60 and K562 cells. The cell death induced by MMS predominantly exhibited the morphological and biochemical hallmarks of necrosis. A minor population of dying cells exhibited apoptotic hallmarks, especially in K562 cell cultures. Cyclosporin A (CsA) was used to modulate the MMS-induced cell death. Our results indicated that CsA did not prevent cells from dying, but changed the mode of death from necrotic to apoptotic. Surprisingly, CsA enhanced oxidative stress and increased the overall number of dead cells. Based on these results, we conclude that the modulatory effect of CsA on MMS-induced cell death might arise from an interference by CsA with mitochondrial metabolism, rather than from inhibition of the MMS efflux mediated by P-glycoprotein.

Topoisomerase II beta levels are a determinant of melphalan-induced DNA crosslinks and sensitivity to cell death.

The role of topoisomerase (topo) II in DNA repair has yet to be fully elucidated. Current evidence suggesting a role for topo II in the repair of DNA damage has been obtained by using in vitro model systems or inferred from correlative data in drug resistant cell lines. In this study we directly examined the role of topo IIalpha and beta in mediating the repair of melphalan-induced crosslinks in cellular DNA. To accomplish this, we used siRNA technology to knock down either topo IIalpha or beta in human chronic myelogenous leukemia K562 and histiocytic lymphoma U937 cell line. Our data demonstrate that topo IIbeta levels, (but not alpha), are a determinant of melphalan-induced crosslinks and sensitivity to melphalan. Furthermore, we show that knocking down topo IIbeta inhibits the repair of melphalan-induced crosslinks in K562 cells. These studies represent the first direct evidence that topo IIbeta participates in the repair of DNA damage induced by an alkylating agent in cellular DNA. Finally, these results suggest non-redundant roles for these two isoforms in mediating repair of DNA crosslinks.

Comparison of allele specific oligonucleotide-polymerase chain reaction and direct sequencing for high throughput screening of ABL kinase domain mutations in chronic myeloid leukemia resistant to imatinib.

To identify a fast and sensitive method for screening for mutations in patients with imatinib- resistant chronic myeloid leukemia (CML), we compared allele specific oligonucleotide- polymerase chain reaction (ASO-PCR) assay with conventional direct sequencing. Among the 68 imatinib resistant CML patients studied, 18 amino acid substitutions were detected in 44 patients by two assays. The sensitivity of ASO-PCR was superior to that of direct sequencing as it could detect one mutant allele in 100 approximately 100,000 wild type sequences. The fastness, simplicity, and sensitivity of ASO-PCR assays will be useful for routine monitoring of mutations, especially for frequently identified mutations.

The effects of saquinavir on imatinib-resistant chronic myelogenous leukemia cell lines.

We evaluated the effect of the human immunodeficiency virus (HIV) protease inhibitor saquinavir on the imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia cell lines. Saquinavir, which is also a proteasome blocker, showed dose- and time-related anti-proliferative activity, particularly on the imatinib-resistant lines and a pro-apoptotic effect. Association with imatinib caused a significant increase of activity.

Pharmacologic mitogen-activated protein/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase inhibitors interact synergistically with STI571 to induce apoptosis in Bcr/Abl-expressing human leukemia cells.

Interactions between the kinase inhibitor STI571 and pharmacological antagonists of the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) cascade have been examined in human myeloid leukemia cells (K562 and LAMA 84) that express the Bcr-Abl kinase. Exposure of K562 cells to concentrations of STI571 that minimally induced apoptosis (e.g., approximately 200 nM) resulted in early suppression (i.e., at 6 h) of p42/44 MAPK phosphorylation followed at later intervals (i.e., > or =24 h) by a marked increase in p42/44 MAPK phosphorylation/activation. Coadministration of a nontoxic concentration of the MEK1/2 inhibitor PD184352 (5 microM) prevented STI571-mediated activation of p42/44 MAPK. Cells exposed to STI571 in combination with PD184352 for 48 h demonstrated a very dramatic increase in mitochondrial dysfunction (e.g., loss of DeltaPsim and cytosolic cytochrome c release) associated with procaspase-3 activation, poly(ADP-ribose) polymerase cleavage, and the appearance of the characteristic morphological features of apoptosis. Similar results were obtained using other pharmacological MEK1/2 inhibitors (e.g., PD 98059 and U0126) as well as another leukemic cell line that expresses Bcr-Abl (e.g., LAMA 84). However, synergistic induction of apoptosis by STI571 and PD184352 was not observed in human myeloid leukemia cells that do not express the Bcr-Abl kinase (e.g., HL-60 and U937) nor in normal human peripheral blood mononuclear cells. Synergistic potentiation of STI571-mediated lethality by PD184352 was associated with multiple perturbations in signaling and apoptotic regulatory pathways, including caspase-dependent down-regulation of Bcr-Abl and Bcl-2; caspase-independent down-regulation of Bcl-x(L) and Mcl-1; activation of JNK, p38 MAPK, and p34(cdc2); and diminished phosphorylation of Stat5 and CREB. Significantly, coexposure to PD184352 strikingly increased the lethality of a pharmacologically achievable concentration of STI571 (i.e., 1-2 microM) in resistant K562 cells expressing marked increases in Bcr-Abl protein levels. Together, these findings raise the possibility that treatment of Bcr-Abl-expressing cells with STI571 elicits a cytoprotective MAPK activation response and that interruption of the latter pathway (e.g., by pharmacological MEK1/2 inhibitors) is associated with a highly synergistic induction of mitochondrial damage and apoptosis. They also indicate that in the case of Bcr-Abl-positive cells, simultaneous interruption of two signal transduction pathways may represent an effective antileukemic strategy.

Beta-hydroxyisovalerylshikonin induces apoptosis in human leukemia cells by inhibiting the activity of a polo-like kinase 1 (PLK1).

beta-Hydroxyisovalerylshikonin (beta-HIVS), which was isolated from the plant, Lithospermum radix, induces apoptosis in various lines of human tumor cells. To identify genes involved in beta-HIVS-induced apoptotic process, we performed cDNA array analysis and found that beta-HIVS suppresses the expression of the gene for a polo-like kinase 1 (PLK1) that is involved in control of the cell cycle. When U937 and HL60 cells were treated with 10(-6) M beta-HIVS for 0.5 h, both the amount of PLK1 itself and the kinase activity of this enzyme were decreased. By contrast, Bcr-Abl-positive K562 cells were resistant to the induction of apoptosis by beta-HIVS and this compound did not suppress the kinase activity of PLK1 in these cells. However, simultaneous treatment of K562 cells with both beta-HIVS and STI571, which selectively inhibits the protein tyrosine kinase (PTK) activity of Bcr-Abl, strongly induced apoptosis. Moreover, beta-HIVS increased the inhibitory effect of STI571 on PTK activity. Treatment of K562 cells with antisense oligodeoxynucleotides (ODNs) specific for PLK1 sensitized these cells to the beta-HIVS-induced fragmentation of DNA. These results suggest that suppression of the activity of PLK1 via inhibition of tyrosine kinase activity by beta-HIVS might play a critical role in the induction of apoptosis.

Selective pyrrolo-pyrimidine inhibitors reveal a necessary role for Src family kinases in Bcr-Abl signal transduction and oncogenesis.

Chronic myelogenous leukemia (CML) is defined by the presence of the Philadelphia (Ph) chromosome, which results in the expression of the 210 kDa Bcr-Abl tyrosine kinase. Bcr-Abl constitutively activates several signaling proteins important for the proliferation and survival of myeloid progenitors, including the Src family kinases Hck and Lyn, the Stat5 transcription factor and upstream components of the Ras/Erk pathway. Recently, we found that kinase-defective Hck blocks Bcr-Abl-induced transformation of DAGM myeloid leukemia cells to cytokine independence, suggesting that activation of the Src kinase family may be essential to oncogenic signaling by Bcr-Abl. To investigate the contribution of Src kinases to Bcr-Abl signaling in vivo, we used the pyrrolo-pyrimidine Src kinase inhibitors PP2 and A-419259. Treatment of the Ph+ CML cell lines K-562 and Meg-01 with either compound resulted in growth arrest and induction of apoptosis, while the Ph- leukemia cell lines TF-1 and HEL were unaffected over the same concentration ranges. Suppression of Ph+ cell growth by PP2 and A-419259 correlated with a decrease in Src kinase autophosphorylation. Both inhibitors blocked Stat5 and Erk activation, consistent with the suppressive effects of the compounds on survival and proliferation. In contrast, the phosphotyrosine content of Bcr-Abl and its endogenous substrate CrkL was unchanged at inhibitor concentrations that induced apoptosis, blocked oncogenic signaling and inhibited Src kinases. These data implicate the Src kinase family in Stat5 and Erk activation downstream of Bcr-Abl, and identify myeloid-specific Src kinases as potential drug targets in CML.

Ectopic expression of c-myc fails to overcome downregulation of telomerase activity induced by herbimycin A, but ectopic hTERT expression overcomes it.

Telomerase plays a key role in the maintenance of chromosomal stability in tumors, but the mechanism regulating telomerase activity is still unclear. Recent studies have suggested that c-myc may be vital for regulation of hTERT mRNA expression and telomerase activity. In this study, we investigated the changes of telomerase activity and telomerase-related genes induced by herbimycin A in K562 human chronic myelogeous leukemic cells. Telomerase activity showed a biphasic pattern in herbimycin A-treated K562 cells. Initially, the telomerase activity decreased along with the decline of cells in S and G2/M phases, but it recovered slightly at the end of treatment. Expression of mRNA for the telomerase catalytic subunit (hTERT) was decreased before the decline of telomerase activity, and increased slightly before the reactivation of telomerase activity. During herbimycin A treatment, both c-myc and cyclin D1 mRNA showed transient downregulation before the increase of G1 cells. Herbimycin A treatment caused the downregulation of both telomerase activity and hTERT mRNA in cyclin D1-transfected K562 cells, while telomerase activity was partially restored in c-Myc-transfected cells. In contrast, hTERT-transfected K562 cells maintained a high level of telomerase activity during herbimycin A treatment. Neither the template RNA component of telomerase (hTERC) nor telomerase-associated protein (TEP-1) were altered in any of the transfected K562 cells. These results indicate that telomerase activity is mainly regulated by hTERT, and that c-Myc protein is one of the positive regulators of hTERT in leukemic cells but is not enough to counteract the downregulation of telomerase activity by herbimycin A completely.

Quantitation of DNA topoisomerase IIalpha and beta in human leukaemia cells by immunoblotting.

DNA topoisomerase II (topo II) is an essential nuclear enzyme and is the target for etoposide, which is used in the therapy of childhood acute lymphoblastic leukaemia (ALL). Topo II exists as two isoforms referred to as topo IIalpha and topo IIbeta. To determine whether cellular levels of topo IIalpha and beta are an important factor in determining drug sensitivity/resistance requires accurate, precise measurements of the two isoforms. We have developed a quantitative Western blotting method to accurately measure the absolute amounts of human topo IIalpha and beta, using recombinant human topo IIalpha and beta as standards. This quantitative method has been used to assess the efficiency of several commonly used topo II extraction protocols. The extractable amount of topo IIalpha and beta was found to be salt-dependent. However extraction using the optimal salt concentration was found to be as efficient as extraction with DNase I/Rnase A digestion and SDS solubilisation. Using the optimum extraction procedure and the quantitative immunoblotting method, topo IIalpha and beta was quantified in cell lines, peripheral blood lymphocytes and in lymphoblasts from children with newly diagnosed ALL.

Activation of caspases and induction of apoptosis by novel ribonucleotide reductase inhibitors amidox and didox.

OBJECTIVE: Amidox and didox are two polyhydroxy-substituted benzohydroxamic acid derivatives that belong to a new class of ribonucleotide reductase (RR) inhibitors. RR is the rate-limiting enzyme for de novo deoxyribonucleotide synthesis, and its activity is significantly increased in tumor cells in proportion to the proliferation rate. Therefore, RR is a target for antitumor therapy. MATERIALS AND METHODS: HL-60 and K562 leukemia cells were treated with increasing doses of amidox and didox. Thereafter, the mode of cytotoxic drug action was determined by Hoechst 33258/propidium iodide (HO/PI) double staining, annexin binding, DNA fragmentation, and caspase activation. This was correlated to the decrease in dNTP levels. Staining with HO/PI and binding of fluorescein isothiocyanate-conjugated annexin V to externalized phosphatidylserine were used to quantify apoptosis. RESULTS: Low doses of amidox or didox resulted in an increase of apoptotic HL-60 cells within 48 hours. Higher doses (50 microM amidox or 250 microM didox) led to rapid induction of apoptosis, which could be detected as early as 4 hours after treatment. After 48 hours with these concentrations, almost 100% of the HL-60 cells died by apoptosis without an increase in necrosis. K562 cells were found to be resistant to amidox but not to didox. In HL-60 cells, upstream caspase 8 is processed in response to didox, whereas caspases 8 and 9 are processed upon amidox treatment. Didox-induced apoptosis, but not amidox-induced apoptosis, can be correlated with the decrease in dNTP levels. The results suggests that amidox induces several apoptosis mechanisms in HL-60 cells. In contrast, only caspase 9 is activated by didox in K562 cells, and because amidox hardly induces apoptosis in this cell line, no caspase cleavage is observed. CONCLUSIONS: Didox triggers distinct apoptosis pathways in HL-60 and K562 cells.

Isolation and characterization of human nuclear and cytosolic multisynthetase complexes and the intracellular distribution of p43/EMAPII.

In this study, the human multienzyme aminoacyl-tRNA synthetase "core" complex has been isolated from the nuclear and cytosolic compartments of human cells and purified to near homogeneity. It is clear from the polypeptide compositions, stoichiometries, and three-dimensional structures that the cytosolic and nuclear particles are very similar to each other and to the particle obtained from rabbit reticulocytes. The most significant difference observed via aminoacylation activity assays and densitometric analysis of electrophoretic band patterns is a lower amount of glutaminyl-tRNA synthetase in the human particles. However, this is not enough to cause major changes in the three-dimensional structures calculated from samples negatively stained with either uranyl acetate or methylamine vanadate. Indeed, the latter samples produce volumes that are highly similar to an initial structure previously calculated from a frozen hydrated sample of the rabbit multisynthetase complex. New structures in this study reveal that the three major structural domains have discrete subsections. This information is an important step toward determination of specific protein interactions and arrangements within the multisynthetase core complex and understanding of the particle's cellular function(s). Finally, gel filtration and immunoblot analysis demonstrate that a major biological role for the cytokine precursor p43 is as an integral part of the multisynthetase complex.

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.

HL-60 cell growth-conditioned medium is an effective inducer of myeloperoxidase expression in K-562 human leukemia cells.

K-562 cells were cultured in HL-60 cell growth-conditioned medium (GCM) for up to 96h. Myeloperoxidase (MPO) mRNA was transiently detected by reverse transcription-polymerase chain reaction (RT-PCR) techniques at 12, 24, and 48h. The de novo expression of MPO protein was subsequently detectable by intracellular flow cytometry at 24, 48, 72 and 96h. Immunogold staining and cytochemical analysis demonstrated granularly-sequestered MPO in approximately 40% of HL-60 GCM-cultured cells after 48h of culture. The sequential detection of MPO mRNA and MPO biosynthesis is considered an indicator of serial maturation evocative of myeloblastic cells, and suggest that K-562 cells maintain the ability to differentiate along this lineage.

A novel method for single cell detection of in situ telomerase or histone H3 in combination with clonal analysis by FISH.

A novel method for simultaneously detecting clonality by FISH, and presence of telomerase activity (telo+ cells) or histone H3 mRNA (H3+) in single cells from a mixed leukemic population is reported. The methods were validated using K562 cells mixed with peripheral blood granulocytes and bone marrow aspirate cells from newly diagnosed AML patients. Fifty patients with AML were analyzed for telo+ cells, while eight AML patients were analyzed for FISH-Telomerase and FISH-H3+ during remission induction therapy. Our results demonstrate that: (1). changes in the leukemic populations during therapy could be followed; (2). a favorable response to chemotherapy occurred when there was a reduction in both the cytogenetically abnormal cells along with reduction in telo+ cells within this abnormal population; (3). reduction of either telo+ cells or FISH+ cells alone did not correlate with good response. H3+ could be detected in only 4% of the leukemic population, most of which were cytogenetically abnormal. These newly established methods allow sub-populations of cells to be followed during disease progression and treatment and to elucidate factors that give a specific clone proliferative advantage.

Coamplification of 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in methotrexate-resistant human leukemia cell lines.

Methotrexate (MTX)-resistant K562 human myelocytic leukemia sublines with 20- and 200-fold amplified dihydrofolate reductase (DHFR) genes localized to homogeneously staining regions (HSRs) on the long arms of chromosomes 5, 6, and 19 were used to examine whether other genes mapping close to the DHFR genes were coamplified. The gene for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, located on chromosome 5q13.3-14, was coamplified 4-14-fold, corresponding to the levels of resistance exhibited by these cells. Similar observations were made with a MTX-resistant subline of the promyelocytic leukemia cell line, HL-60R, with 200 gene copies of DHFR. These observations indicate a tight linkage of DHFR and HMG-CoA genes on chromosome 5q.

Mechanism of beta 2-microglobulin-induced apoptosis in the K562 leukemia cell line, defective in major histocompatibility class 1.

BACKGROUND: It has been shown that exogenous beta 2-microglobulin (beta 2m) triggers significant apoptosis in several cell lines, but the molecular mechanism of beta 2m-induced apoptosis remains to be found. MATERIALS AND METHODS: To understand the mechanism of beta 2m-induced apoptosis, we added purified human beta 2m to cultures of K562 human chronic myelocytic leukemia cells, detected apoptosis by DNA fragmentation and annexin V binding assays, measured mitochondrial membrane potential (delta psi m), and used Z-VAD-fmk, a general inhibitor of caspases, inhibitors of caspases-1 and-3, as well as Western blot analysis to detect activated caspases. RESULTS: beta 2m-induced apoptosis was associated with decreased delta psi m K562 cells. Treatment with the general caspase inhibitor Z-VAD-fmk, as well as the caspase-1 inhibitor YVAD-CHO, significantly blocked beta 2m-induced apoptosis. However, Western blot analysis revealed that caspase-1 was intrinsically activated in untreated as well as beta 2m-treated K562 cells. Furthermore, beta 2m-induced apoptosis was not associated with the cleavage of caspase-3 as revealed by Western blot analysis, but was inhibited by an inhibitor of caspase-3, Z-DEVD-CHO, suggesting that not caspase-3, but a caspase-3-like enzyme may be involved in beta 2m-induced apoptosis. Western blot analysis also revealed that caspases-4, -8 and -9 were not activated during beta 2m-induced apoptosis in these cells. CONCLUSION: These results reveal that beta 2m-induced apoptosis in K562 cells occurs by a mechanism dependent on decreased mitochondrial delta psi m. Moreover, while caspase-1 activity may be one of the factors involved in beta 2m-induced apoptosis, activation of this caspase alone does not cause apoptosis, and other proapoptotic factors including activation of a caspase-3-like enzyme, independently of caspases-4, -8 and -9 activity, may be required to trigger apoptosis.