Human Lymphoblastic Leukemia Cell Line Research Articles

Alterations in γ-Actin and Tubulin-Targeted Drug Resistance in Childhood Leukemia

Proteomic investigations have revealed alterations in cytoskeletal proteins expressed in human acute lymphoblastic leukemia cells that are resistant to microtubule-disrupting agents. We characterized gamma-actin expression in antimicrotubule drug-resistant leukemia and examined the effect of altered gamma-actin in resistance of acute lymphoblastic leukemia to antimicrotubule agents. Two-dimensional polyacrylamide gel electrophoresis and mass spectrometry were used to identify actin proteins in human acute lymphoblastic leukemia cell lines resistant to vinblastine (CCRF-CEM/VLB100 cells) and desoxyepothilone B (CCRF-CEM/dEpoB140 cells). Fluorescence-based cycle sequencing was used to detect gene mutations. Site-directed mutagenesis was used to generate mutant gamma-actin expression plasmids, which were used to transfect mouse NIH/3T3 cells. Clonogenic analysis was used for drug sensitivity studies. A small interfering RNA (siRNA) was used to block gamma-actin gene expression in human neuroblastoma SH-EP cells. Expression of gamma-actin (normalized to that of beta2-microglobulin [beta2M]) in primary leukemia cells obtained from patients at diagnosis (n = 44) and relapse (n = 25) was examined using semiquantitative reverse transcription-polymerase chain reaction. Statistical significance of changes in the ratio of gamma-actin to beta2M expression between diagnosis and relapse samples was determined by two-sided unpaired Student's t tests. We identified novel mutant forms of gamma-actin and the concomitant loss of wild-type gamma-actin in CCRF-CEM/VLB100 cells and CCRF-CEM/dEpoB140 cells. Mouse NIH/3T3 cells that expressed the mutant gamma-actin proteins were more resistant to antimicrotubule agents than cells transfected with empty plasmid. Human neuroblastoma SH-EP cells transfected with gamma-actin siRNA displayed higher relative resistance to paclitaxel (P<.001), vinblastine (P = .04), and epothilone B (P = .045) than mock-transfected cells. No gamma-actin gene mutations were identified in 37 samples of primary leukemia cells (eight from patients at diagnosis, 29 from patients at relapse). Gamma-actin gene expression was lower in acute lymphoblastic leukemia samples collected at clinical relapse (n = 25; mean gamma-actin/beta2M = 0.53) than in samples collected at diagnosis (n = 44; mean gamma-actin/beta2M = 0.68; difference = 0.15, 95% confidence interval [CI] = 0.04 to 0.27, P = .01). These data provide functional and associative clinical evidence of a novel form of drug resistance that involves interactions between gamma-actin and microtubules.

Lipid raft disruption prevents apoptosis induced by 2-chloro-2′-deoxyadenosine (Cladribine) in leukemia cell lines

To clarify the role of lipid rafts in 2-chloro-2′-deoxyadenosine (2CdA; Cladribine)-induced apoptosis, the effects of disruption of lipid rafts by methyl-β-cyclodextrin (MβCD) and filipin on 2CdA-induced apoptosis were investigated in four human acute lymphoblastic leukemia (ALL) cell lines comprised of T cells (MOLT-4, Jurkat) and B cells (NALM, BALL-1). The disruption of lipid rafts significantly inhibited 2CdA-induced apoptosis, indicating the crucial role of lipid rafts in the induction of apoptosis in leukemia cells. These reagents significantly inhibited 2CdA-induced elevation of the intracellular calcium concentration ([Ca 2+] i ) in MOLT-4 cells, and 2CdA-induced apoptosis was partly inhibited by the Ca 2+ chelators BAPTA-AM and EGTA, and the L-type Ca 2+ channel blocker nifedipine. On the other hand, they had no effects on the cellular uptake of 2CdA. These results indicated that lipid rafts partly contributed to 2CdA-induced apoptosis by regulating Ca 2+ influx via the plasma membrane.

Growth inhibition and apoptosis in human Philadelphia chromosome-positive lymphoblastic leukemia cell lines by treatment with the dual PPARα/γ ligand TZD18

Treatment of adult Philadelphia chromosome-positive lymphocytic leukemia is rarely successful. We report here the effects of TZD18, a novel dual ligand specific for peroxisome proliferator-activated receptor alpha and gamma (PPARalpha/gamma) on Ph(+) lymphocytic leukemia cell lines BV173, SD1, and SupB-15. Exposure of these cells to TZD18 resulted in growth inhibition in a dose- and time-dependent manner that was associated with G(1) cell cycle arrest. This effect was much stronger than that mediated by the PPARgamma ligand pioglitazone (PGZ), which also belongs to the thiazolidinediones (TZD) class of ligands. However, it may not be mediated through PPARgamma or PPARalpha activation because antagonists of PPARgamma and PPARalpha cannot reverse it. Study of the key regulators of cell cycle progression by Western blot analysis showed that the expression of the cyclin-dependent kinase inhibitor (CDKI) p27(kip1), but not that of p21(cip1), was enhanced, whereas that of c-Myc, cyclin E, cyclin D2, and cyclin-dependent kinases 2 and 4 (CDK-2 and CDK-4) was decreased when these cells were treated with TZD18 (10 or 20 microM). Therefore, the up-regulation of p27(kip1) and the down-regulation of CDK-2 and CDK-4 may, at least in part, account for the G(1) cell cycle arrest. Furthermore, a remarkable induction of apoptosis was observed in the cells treated with this dual ligand. No obvious alteration of bcl-2 protein level occurred, but bax was up-regulated in these TZD18-treated cells. Activation of caspase 8 and caspase 9 by TZD18 was also observed. Importantly, NF-kappaB DNA-binding activity was markedly decreased by the TZD18 treatment. In addition, TZD18 enhanced the growth inhibitory effect of imatinib, a specific tyrosine kinase inhibitor therapeutically used in the treatment of Ph(+) leukemia. Overall, our findings strongly suggest that TZD18 may offer a new therapeutic approach to aid in the treatment of Ph(+) lymphocytic leukemia.

Cellular FLICE-like inhibitory protein (c-FLIP): A novel target for Taxol-induced apoptosis

It is known that by binding to the FAS-associated death domain (FADD) protein and/or caspases-8 and -10 at the level of the death-inducing signaling complex (DISC), cellular FLICE-like inhibitory protein (c-FLIP) can prevent apoptosis triggered by death-inducing ligands. We investigated whether the c-FLIP splice variants, c-FLIP long [c-FLIP(L)] and c-FLIP short [c-FLIP(S)], play a role in Taxol-induced apoptosis. Our results showed that low Taxol concentrations triggered caspase-8- and caspase-10-dependent apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line, and induced the down-regulation of c-FLIP(S) and c-FLIP(L). Taxol decreased the expression of c-FLIP by a post-transcriptional and caspase-independent mechanism. To explore the distinct functions of the c-FLIP variants in Taxol-induced apoptosis, we transfected the cells with expression vectors carrying c-FLIP(L) and c-FLIP(S) in the sense orientation or c-FLIP(S) in the antisense orientation [c-FLIP(S)-AS]. Caspases-8 and -10 were more efficiently activated in the c-FLIP(S)-AS strain treated with 5–50 nM Taxol, which revealed that c-FLIP regulates Taxol-induced apoptosis by interacting with these caspases. Furthermore, our data showed that increased expression of c-FLIP(L) or c-FLIP(S) reduced apoptosis at 5–50 nM Taxol concentrations suggesting that both isoforms of c-FLIP prevent Taxol-induced apoptosis. These results revealed that Taxol induces apoptosis by down-regulating c-FLIP(S) and c-FLIP(L) expression.

Apoptosis Induction in Human Lymphoma and Leukemia Cell Lines by Transfection via Dendrosomes Carrying Wild-Type p53 cDNA

The efficiency of dendrosomes (novel dendritic spheroidal nanoparticle gene porters) were assessed in transferring wild-type p53 cDNA into two human acute lymphoblastic leukemia cell lines (MOLT-4 and CCRF-CEM) derived from T cells and erythroleukemic cell line K562. Flow cytometric studies showed a 65% and 45% enhancement in apoptosis and necrosis of K562 and CCRF-CEM cells transfected with complex of dendrosomes and wild-type p53 cDNA in comparison to controls. The cytotoxicity studies on T lymphoma cells revealed that dendrosomes have a low cytotoxicity in comparison to lipofectin.

The Activity of Combined Topoisomerase Inhibitors Is Augmented by Octreotide in the Treatment of Lymphoid Leukemias.

Clinical protocols combining a topoisomerase I (topo I) and a topoisomerase IIalpha (topo II inhibitor) have shown major responses against several tumours including acute leukemia and myelodysplastic syndromes. However, combinations with topoisomerase targeting drugs should be considered with caution because antagonistic effects have been observed when administering camptothecin or topotecan (topo I inhibitors) with doxorubicin (topo II inhibitor). Octreotide (OCT) is an eight amino-acid peptide, which attains its biological effects on target cells by binding preferentially to sst2 and, to a lesser extent, to sst3 and sst5 somatostatin receptors (SS-Rs). The presence of SS-Rs in human lymphoid leukemia cell lines, in malignant lymphomas and in lymphoproliferative diseases is clearly detected. We studied the in vitro effect of combinations of OCT with doxorubicin (DOX) and topotecan (TP) on cell growth and viability in four human lymphoblastic leukemia cell lines (CCRF-CEM, RPMI-8226, JURKAT, MOLT-4) and on the Topo I, IIalpha and sst2 expression in JURKAT, MOLT-4 leukemia cell lines, as well as the in vivo effect on rodent P388 lymphocytic and L1210 lymphoid leukemias. In vitro growth inhibition and cytotoxicity were evaluated with the MTT colormetric metabolic assay. Topo I, IIalpha and sst2 mRNAs were detected with RT-PCR and the quantification of the electrophoresed specific PCR products was accomplished with Molecular Imager FX. In vivo antitumour activity was estimated by the % survival ratio of treated to untreated (control) mice and the ratio of 70-day tumour free survivors (cures). The in vitro growth inhibition and cytotoxicity that were induced by the DOX and TP combinations were neither synergistic nor additive and were similar to the activity of DOX alone. However, with the addition of OCT to the DOX and TP combinations a significant (p<0.001) synergistic effect was resulted in all tested cell lines. Treatment of cell lines with DOX produced almost a total consumption of Topo I and IIalpha mRNAs and with TP induced increase of Topo IIalpha mRNA levels (0.5–1.5 folds). OCT is clearly upregulates the Topo IIalpha expression (>2.5 folds) and restores Topo I mRNA production in cells treated with DOX. The sst2 mRNA levels were not affected in any case. In vivo antitumour activity of DOX and TP combinations was neither synergistic nor additive and it was similar to the activity of DOX alone. The addition of OCT to DOX and TP combinations produced an important synergistic antitumour effect increasing significantly survival time and cures (p<0.01) in both P388 and L1210 leukemias. Our data indicate that the antagonistic effects of Topo I and Topo II inhibitors may be due to effects on the regulation of topoisomerases expression. OCT significantly enhances the antileukemic activity of combinations with such important anticancer drugs, upregulating Topo I and IIalpha expression.

Evaluation of glucocorticoid sensitivity in 697 pre-B acute lymphoblastic leukemia cells after overexpression or silencing of MAP kinase phosphatase-1

To determine the effect of modulating MAP kinase phosphatase-1 (MKP-1) expression levels on cell death induced by glucocorticoid (GC) or hydroxyurea (HU) treatment in the human pre-B acute lymphoblastic leukemia cell line 697. Stable MKP-1 overexpressing transformants of the 697 pre-B acute lymphoblastic leukemia cell line were created and tested for sensitivity to the GC triamcinolone acetonide (TA) and HU, and compared to a control 697 cell line containing normal MKP-1 expression levels. Small interfering RNAs (siRNAs) were designed to inhibit MKP-1 expression and evaluated for their effect on GC-mediated cell death. MKP-1 overexpression caused a phenotype of partial resistance to HU-induced apoptosis but not to GC-induced apoptosis. Electroporation of siRNAs effectively silenced MKP-1 expression, and increased sensitivity to TA by 9.6+/-1.9%. Because MKP-1 protects certain tumor cells from chemotherapy-induced apoptosis, its inhibition is being considered as a possible strategy for combination cancer therapy. However, this study suggests that while MKP-1 inhibition may improve the efficacy of DNA damaging agents, it may have only limited utility in combination with glucocorticoids.

Taxol Induces Caspase-10-dependent Apoptosis

Taxol (paclitaxel) is known to inhibit cell growth and trigger significant apoptosis in various cancer cells. Although taxol induces apoptosis of cancer cells, its exact mechanism of action is not yet known. In this study we investigated death receptors, FAS-associated death domain protein (FADD), the activation of caspases-10 and -8 as well as the downstream caspases, and reactive oxygen species (ROS) in taxol-induced apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line. Pretreating the cells with neutralizing antibodies to Fas, tumor necrosis factor (TNF)-alpha receptor 1, or TNF-related apoptosis-inducing ligand receptors (DR4 and DR5) did not affect taxol-induced apoptosis, but transfection of the cells with a dominant negative FADD plasmid resulted in inhibition of taxol-induced apoptosis, revealing that taxol induces apoptosis independently of these death receptors but dependently on FADD. Furthermore, the drug induced activation of caspases-10, -8, -6, and -3, cleaved Bcl-2, Bid, poly(ADP-ribose) polymerase, and lamin B, and down-regulated cellular levels of FLICE-like inhibitory protein (FLIP) and X-chromosome-linked inhibitor of apoptosis protein (XIAP). However, despite the release of cytochrome c from the mitochondria in taxol-treated cells, caspase-9 was not activated. Inhibitors of caspases-8, -6, or -3 partially inhibited taxol-induced apoptosis, whereas the caspase-10 inhibitor totally abrogated this process. Taxol-induced apoptosis was also associated with decreased mitochondrial membrane potential (Deltapsim) and a significant increase in ROS generation. However, increased ROS production was not directly involved in taxol-triggered apoptosis. Therefore, these results demonstrate for the first time that taxol induces FADD-dependent apoptosis primarily through activation of caspase-10 but independently of death receptors.

Cyp33-MLL Interaction Modulates Human HOX Gene Expression and Chromatin Structure.

The MLL gene is involved in translocations and fusion genes associated with human leukemia. We have identified a protein, Cyp33, which interacts with the PHD fingers of MLL, and modulates the expression of HOX genes regulated by MLL. In order to define possible mechanisms for this modulation, we set up an experimental system in which the Cyp33 protein is conditionally expressed from a transgene under the control of a tetracycline inducible promoter. Two different cell lines with such inducible gene were established, based on the human kidney epithelial cells HEK293, and the human lymphoblastic leukemia cell line Jurkat, respectively. In these systems we have shown that induction of Cyp33 expression over endogenous levels, leads to downregulation of the HOXC8 gene, but this downregulation is reversible, the HOXC8 being re-expressed when Cyp33 is de-induced and its levels go back to normal. This indicates that Cyp33 overexpression does not erase an epigenetic mark for HOXC8 gene expression. Concordantly, no change in methylation of histone H3 at lysines 4 or 9 is observed upon Cyp33 overexpression. We also showed that both MLL and Cyp33 bind to the promoter of HOXC8, and that histones H3 and H4 at this promoter are de-acetylated upon Cyp33 overexpression. This deacetylation, as well as the downregulation of HOXC8 expression, are prevented by cyclosporin A and by trichostatin A, suggesting that the prolyl-isomerase activity of Cyp33 is necessary for these effects, and that histone deacetylation mediates these effects. We have previously shown that overexpression of Cyp33 enhances the recruitment of HDAC1 to the repression domain of MLL. We now show that this enhanced recruitment is inhibited by cyclosporin A. These results suggest a model in which Cyp33 is recruited to MLL and targets the MLL repression domain to promote the recruitment of histone deacetylases, thus allowing or promoting the silencing of the MLL regulated genes. In this model Cyp33 functions downstream of the gene activation or silencing epigenetic marks.

202 The proteasome inhibitor bortezomib (VELCADE™) sensitizes human tumor cells to TRAIL-mediated apoptosis by reduction of c-FLIP

It is known that by binding to the FAS-associated death domain (FADD) protein and/or caspases-8 and -10 at the level of the death-inducing signaling complex (DISC), cellular FLICE-like inhibitory protein (c-FLIP) can prevent apoptosis triggered by death-inducing ligands. We investigated whether the c-FLIP splice variants, c-FLIP long [c-FLIP(L)] and c-FLIP short [c-FLIP(S)], play a role in Taxol-induced apoptosis. Our results showed that low Taxol concentrations triggered caspase-8- and caspase-10-dependent apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line, and induced the down-regulation of c-FLIP(S) and c-FLIP(L). Taxol decreased the expression of c-FLIP by a post-transcriptional and caspase-independent mechanism. To explore the distinct functions of the c-FLIP variants in Taxol-induced apoptosis, we transfected the cells with expression vectors carrying c-FLIP(L) and c-FLIP(S) in the sense orientation or c-FLIP(S) in the antisense orientation [c-FLIP(S)-AS]. Caspases-8 and -10 were more efficiently activated in the c-FLIP(S)-AS strain treated with 5–50 nM Taxol, which revealed that c-FLIP regulates Taxol-induced apoptosis by interacting with these caspases. Furthermore, our data showed that increased expression of c-FLIP(L) or c-FLIP(S) reduced apoptosis at 5–50 nM Taxol concentrations suggesting that both isoforms of c-FLIP prevent Taxol-induced apoptosis. These results revealed that Taxol induces apoptosis by down-regulating c-FLIP(S) and c-FLIP(L) expression.

Apoptosis of human T-cell acute lymphoblastic leukemia cells by diphenhydramine, an H1 histamine receptor antagonist.

Recently, it has been demonstrated that histamine plays an important role in the proliferation of normal and malignant cells. We have examined the effects of histamine, diphenhydramine, and cimetidine (H1 and H2 histamine receptor antagonists, respectively) on the in vitro proliferation of two human T-cell acute lymphoblastic leukemia cell lines, namely CCRF-CEM and Jurkat. Exogenous histamine did not alter the proliferation or viability of these cells. In contrast, diphenhydramine induced apoptosis in a dose- and time-dependent manner in both cell lines, whereas cimetidine failed to induce significant effects at similar concentrations. Diphenhydramine-induced apoptosis was evaluated in terms of morphology, flow cytometry, and the release of cytochrome c to the cytosol. The latter was partially mitigated by Bcl-2 overexpression. In human peripheral blood mononuclear cells, diphenhydramine inhibited cell proliferation without inducing apoptosis. Our findings indicate that endogenous histamine may be an important factor for the survival of CCRF-CEM, Jurkat, and peripheral blood mononuclear cells, and point to the potential application of H1 receptor antagonists as cytotoxic agents for the specific treatment of certain types of leukemia.

Altered conjugate formation and altered apoptosis of multidrug-resistant human leukemia cell line affects susceptibility to killing by activated natural killer (NK) cells.

Most leukemias that exhibit P-glycoprotein (P-gp)-associated multidrug resistance (MDR) exhibit reduced susceptibility to immune cytotoxicity mediated by natural killer (NK) cells. To explore this phenomenon we investigated N6/ADR, a doxorubicin-selected, P-gp-positive variant of the human acute lymphoblastic leukemia (ALL) cell line NALM6. Each stage of the NK cytolytic pathway, (binding, activation and killing) was evaluated to identify the alterations responsible for the reduced cytotoxicity of the variant relative to its drug-sensitive parental line. The major cause of the decreased susceptibility to NK cytolysis was found to be reduced conjugate formation by the MDR variant. Activation of NK effectors by parental and MDR cells with concomitant release of tumor necrosis factor-alpha (TNF-alpha) correlated with conjugate formation. N6/ADR was also more resistant than NALM6 to antibody-dependent cellular cytotoxicity and to cytotoxic factors released from NK cells as measured both by 51Cr-release and by DNA fragmentation. This is the first report of a P-gp-positive leukemic line that exhibits reduced conjugate formation as well as increased resistance to NK-mediated killing mechanisms. Our results suggest caution in the use of NK-based immunotherapy as an alternative treatment for multidrug-resistant leukemias.

Association of Transcription Factor YY1 with the High Molecular Weight Notch Complex Suppresses the Transactivation Activity of Notch

Notch receptors are evolutionarily conserved from Drosophila to human and play important roles in cell fate decisions. After ligand binding, Notch receptors are cleaved to release their intracellular domains. The intracellular domains, the activated form of Notch receptors, are then translocated into the nucleus where they interact with other transcriptional machinery to regulate the expression of cellular genes. To dissect the molecular mechanisms of Notch signaling, the cellular targets that interact with Notch1 receptor intracellular domain (N1IC) were screened. In this study, we found that endogenous transcription factor Ying Yang 1 (YY1) was associated with exogenous N1IC in human K562 erythroleukemic cells. The ankyrin (ANK) domain of N1IC and zinc finger domains of YY1 were essential for the association of N1IC and YY1 according to the pull-down assay of glutathione S-transferase fusion proteins. Furthermore, both YY1 and N1IC were present in a large complex of the nucleus to suppress the luciferase reporter activity transactivated by Notch signaling. The transcription factor YY1 indirectly regulated the transcriptional activity of the wild-type CBF1-response elements via the direct interaction of N1IC and CBF1. We also demonstrated the association between endogenous N1IC and intrinsic YY1 in human acute T-cell lymphoblastic leukemia cell lines. Taken together, these results indicate that transcription factor YY1 may modulate Notch signaling via association with the high molecular weight Notch complex.

Dose-dependent transcriptome changes by metal ores on a human acute lymphoblastic leukemia cell line

The increased morbidity of childhood leukemia in Fallon, Nevada and Sierra Vista, Arizona has prompted great health concern. The main characteristic that these two towns share is the environmental pollution attributed to metal ore from abandoned mining operations. Consequently, we have investigated the transcriptome effects of metal ores from these endemic areas using a human T-cell acute lymphoblastic leukemia cell line (T-ALL). Metal ore from Fallon significantly increased cell growth after 24, 48 and 72 h of incubation at 1.5 microg/mL concentration, as measured by trypan-blue. Sierra Vista ore significantly increased cell growth with 0.15 and 1.5 microg/mL following 72 h of incubation. From human cDNA microarray, results indicate that in total, eight genes, mostly metallothionein (MT) genes, were up-regulated and 10 genes were down-regulated following treatment of the T-ALL cells with 0.15 and 1.5 microg/mL of metal ores at 72 h, in comparison with untreated cells. Twenty-eight metals of both ores were quantified and their presence may be associated with the cell growth rate and dose-dependent activation of transcriptomes in immature T-cells.

Telomerase inhibition enhances apoptosis in human acute leukemia cells: possibility of antitelomerase therapy.

Telomerase is a ribonucleoprotein enzyme that maintains protective structures at the ends of eukaryotic chromosomes. We examined the impact of telomerase inhibition by the dominant-negative human catalytic subunit of telomerase (DN-hTERT) on the biological features of acute leukemia. We introduced vectors encoding dominant- negative (DN)-hTERT, wild-type (WT)-hTERT, or a control vector expressing only a drug-resistant marker into a telomerase-positive human acute lymphoblastic leukemia cell line, HAL-01. Expression of DN-hTERT dramatically inhibited telomerase activity, leading to apoptotic cell death. Mutant telomerase expression also enhanced daunorubicin-induced apoptosis. Nude mice (n=5 per group) received subcutanous implants of HAL-01 cells expressing the control vector or DN-hTERT or WT-hTERT. Implantation of HAL-01 cells expressing control vector (n=5) rapidly produced tumors, whereas implantation of those expressing DN-hTERT (n=5) did not. Thus, telomerase inhibition both growth of HAL-01 cells in vitro and tumorigenic capacity in vivo. Furthermore, the G-quadruplex-interactive telomerase-specific inhibitor, telomestatin, shortened the telomere length and induced apoptosis in freshly isolated primary acute leukemia cells. These results suggest that antitelomerase therapy may be useful in some acute leukemias in combination with antileukemic agents such as daunorubicin.

Pre-B cell antigen receptor-mediated signal inhibits CD24-induced apoptosis in human pre-B cells.

We previously reported that the cross-linking of cluster of differentiation (CD)24 induces apoptosis in Burkitt's lymphoma cells and that this phenomenon can be enhanced by a B cell Ag receptor (BCR)-mediated signal. In this study, we extend our previous observation and report that CD24 also mediated apoptosis in human precursor-B acute lymphoblastic leukemia cell lines in the pro-B and pre-B stages accompanying activation of multiple caspases. Interestingly, simultaneous cross-linking of pre-BCR clearly inhibited CD24-mediated apoptosis in pre-B cells. We also observed that mitogen-activated protein kinases (MAPKs) were involved in the regulation of this apoptotic process. Pre-BCR cross-linking induced prompt and strong activation of extracellular signal-regulated kinase 1, whereas CD24 cross-linking induced the sustained activation of p38 MAPK, following weak extracellular signal-regulated kinase 1 activation. SC68376, a specific inhibitor of p38 MAPK, inhibited apoptosis induction by CD24 cross-linking, whereas anisomycin, an activator of p38 MAPK, enhanced the apoptosis. In addition, PD98059, a specific inhibitor of MEK-1, enhanced apoptosis induction by CD24 cross-linking and reduced the antiapoptotic effects of pre-BCR cross-linking. Collectively, whether pre-B cells survive or die may be determined by the magnitude of MAPK activation, which is regulated by cell surface molecules. Our findings should be important to understanding the role of CD24-mediated cell signaling in early B cell development.

Beta2-microglobulin induces caspase-dependent apoptosis in the CCRF-HSB-2 human leukemia cell line independently of the caspase-3, -8 and -9 pathways but through increased reactive oxygen species.

Exogenous beta(2)-microglobulin (beta(2)m) induces significant apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line as detected by DNA fragmentation, DAPI staining and annexin V binding assay. beta(2)m treatment induced the release of cytochrome c and apoptosis-inducing factor (AIF) from the mitochondria, but no change in mitochondrial membrane potential (DeltaPsim) was observed during apoptosis, suggesting that cytochrome c may be released through a mechanism independent of mitochondrial permeability transition (MPT) pore formation. Moreover, the beta(2)m-induced release of cytochrome c and AIF from the mitochondria in CCRF-HSB-2 cells was caspase-independent, since Z-VAD-fmk, a general inhibitor of caspases, did not block the release of these factors. However, Z-VAD-fmk treatment significantly blocked beta(2)m-induced apoptosis, while Western blot analysis revealed that caspases-1, -2, -3, -6, -7, -8 and -9 are not activated during beta(2)m-induced apoptosis in these cells. These results collectively indicate that a post-mitochondrial caspase-dependent mechanism is involved in beta(2)m-induced apoptosis. Moreover, beta(2)m significantly enhanced the production of reactive oxygen species (ROS) during 12-48 hr treatment, and beta(2)m-induced apoptosis was almost totally inhibited in cells pre-treated with the antioxidant N-acetylcysteine (NAC), providing evidence that beta(2)m-induced apoptosis in CCRF-HSB-2 cells is ROS-dependent. Therefore, these results reveal that beta(2)m-induced apoptosis in CCRF-HSB-2 cells may occur through an unknown caspase-dependent and ROS-dependent mechanism(s) that is associated with cytochrome c and AIF release from mitochondria, but is independent of the caspase -3, -8 and -9 pathways.

A Novel L3-Acute Lymphoblastic Leukemia Cell Line (BALM-27) Carrying Cytoplasmic Immunoglobulin δ Chain but Lacking Expression of Cell Surface Immunoglobulin Chains

A human acute lymphoblastic leukemia (ALL) cell line, BALM-27, was established from the peripheral blood specimen of a patient with B-cell ALL L3 type (ALL-L3) at diagnosis. As with the original leukemia cells, the established line was negative for all cell surface immunoglobulin (Ig) chains, but carrying only cytoplasmic Ig δ heavy chain. Southern blot analysis of the various Ig chain genes demonstrated homozygous deletion of the J κ gene, germ line configuration of the J and rearrangement of IgJH genes. Cytogenetic analysis of both primary leukemic bone marrow and BALM-27 cells showed the der(8;15)(q10;ql0) chromosomal alteration, in addition to the t(8;22)(q24;q11) abnormality which is highly associated with ALL-L3 and Burkitt's lymphoma. The established cell line BALM-27 represents a rich resource of abundant, accessible, and manipulable cell material for analyzing the unique expression of Ig chain and for investigating the pathogenesis of B-cell malignancy. The scientific significance of BALM-27 lies in (1) the rarity of this type of leukemia cell lines, and (2) its unique chromosomal aberrations.

Cytogenetic characterization of chromosomal rearrangement in a human vinblastine-resistant CEM cell line: use of comparative genomic hybridization and fluorescence in situ hybridization

In order to identify genomic changes associated with drug-resistance acquisition, we performed R-banding karyotyping, fluorescence in situ hybridization, and comparative genomic hybridization to compare a human T-cell lymphoblastic leukemia cell line, CEM-wild type, and a subline with resistance to vinblastine (CEM-VLB) and overexpressing P-glycoprotein. Comparative genomic hybridization analysis showed that the CEM-VLB cell line carried chemoresistance-associated chromosomal abnormalities (amplification of 7q11∼q22, losses of chromosomes 2, 3, 5, 9, 10, and 16, and deletion of 4q13∼qter). Fluorescence in situ hybridization identified an amplified 7q21 region translocated on the short arm of a chromosome 2. This region contained the MDR1 gene locus and probably neighboring genes, such as SRI or MDR3/ABCB4. According to previous reports, this chromosomal rearrangement occurred during drug selection and attested a resistance acquisition.

Expression of a Unique Helios Isoform in Human Leukemia Cells

The purpose of the present study was to characterize the human Helios gene products expressed in leukemia cells. A 3.5 kb human Helios cDNA clone was isolated from a human T-cell cDNA library derived from the human T-acute lymphoblastic leukemia (ALL) cell line JURKAT. This cDNA clone had a unique open reading frame (ORF) encoding a novel 304 amino acid (AA) peptide, which was designated as Helios 3. The sequence of the 289 AA C-terminal portion of Helios 3 downstream of V-16 is identical to the corresponding sequence found in Helios 1 and 2 and contains two zinc fingers. By contrast, the 15 AA N-terminal portion of Helios 3 is unique and does not contain the N-terminal zinc finger motifs that are conserved in Helios 1 and 2 as well as other previously identified members of the Ikaros family. Southern blot analysis of genomic DNA fragments of the human Helios gene locus showed that Helios 3 is encoded by the same genomic locus as Helios 1 and 2. The expression of Helios 3 mRNA was not restricted to T-lineage ALL cells or another immunophenotypically distinct subset of leukemias. Helios 3 mRNA was expressed in freshly obtained primary leukemic cells from six of 15 children with newly diagnosed ALL. Helios 3 exhibited a unique protein interaction profile via its N-terminal portion, which may have biological significance in pathogenesis of human leukemias.