Lymphoid Leukemia Cells Research Articles

Gene networks in glucocorticoid-evoked apoptosis of leukemic cells

To discover the genes responsible for the apoptosis evoked by glucocorticoids in leukemic lymphoid cells, we have begun gene array analysis on microchips. Three clones of CEM cells were compared: C7–14, C1–15 and C1–6. C7–14 and C1–15 are subclones from the original clones C7 (sensitive to apoptosis by glucocorticoids) and C1 (resistant). C1–6 is a spontaneous revertant to sensitivity from the C1 clone. Previously we presented data on the sets of genes whose expression is altered in these cell clones after 20 h exposure to dexamethasone (Dex). The two sensitive clones, which respond by undergoing apoptosis starting about 24 h after Dex is added, both showed >2.5-fold induction of 39 genes and 2-fold reduction of expressed levels from 21 genes. C1–15, the resistant clone, showed alterations in a separate set of genes. In this paper, we present further analysis of the data on genes regulated in these cell clones after 20 h Dex and compare them with the genes regulated after 12 h Dex. Some, but not all the genes found altered at 20 h are altered at 12 h, consistent with our hypothesis that sequential gene regulation eventually provokes full apoptosis. We also compare the levels of basal gene expression in the three clones. At the basal level no single gene stands out, but small sets of genes differ >2-fold in basal expression between the two sensitive and the resistant clone. A number of the genes basally higher in the resistant clone are potentially anti-apoptotic. This is consistent with our hypothesis that the resistant cells have undergone a general shift in gene expression.

Treatment-specific changes in gene expression discriminate in vivo drug response in human leukemia cells.

To elucidate the genomics of cellular responses to cancer treatment, we analyzed the expression of over 9,600 human genes in acute lymphoblastic leukemia cells before and after in vivo treatment with methotrexate and mercaptopurine given alone or in combination. Based on changes in gene expression, we identified 124 genes that accurately discriminated among the four treatments. Discriminating genes included those involved in apoptosis, mismatch repair, cell cycle control and stress response. Only 14% of genes that changed when these medications were given as single agents also changed when they were given together. These data indicate that lymphoid leukemia cells of different molecular subtypes share common pathways of genomic response to the same treatment, that changes in gene expression are treatment-specific and that gene expression can illuminate differences in cellular response to drug combinations versus single agents.

Gene expression profile of human lymphoid CEM cells sensitive and resistant to glucocorticoid-evoked apoptosis

Three closely related clones of leukemic lymphoid CEM cells were compared for their gene expression responses to the glucocorticoid dexamethasone (Dex). All three contained receptors for Dex, but only two responded by undergoing apoptosis. After a time of exposure to Dex that ended late in the interval preceding onset of apoptosis, gene microarray analyses were carried out. The results indicate that the expression of a limited, distinctive set of genes was altered in the two apoptosis-prone clones, not in the resistant clone. That clone showed altered expression of different sets of genes, suggesting that a molecular switch converted patterns of gene expression between the two phenotypes: apoptosis-prone and apoptosis-resistant. The results are consistent with the hypothesis that altered expression of a distinctive network of genes after glucocorticoid administration ultimately triggers apoptosis of leukemic lymphoid cells. The altered genes identified provide new foci for study of their role in cell death.

Multidrug resistance modulators PSC 833 and CsA show differential capacity to induce apoptosis in lymphoid leukemia cell lines independently of their MDR phenotype

Among the mechanisms that induce multidrug resistance (MDR), one of those most frequent is over-expression of a phosphoglycoprotein (Pgp) encoded in the mouse by the mdr-1 and mdr-3 genes. We have demonstrated that cyclosporin-A (CsA) as well as its analogue PSC 833 were able to revert the MDR phenotype in murine cell lines resistant to vincristine (LBR-V160) or doxorubicin (LBR-D160). The aim of this work was to evaluate the ability of PSC 833 and CsA to modulate mdr-1, mdr-3 and mrp-1 genes as well as to induce apoptosis analyzing the mechanism involved in the above tumor cell lines. By semi-quantitative RT-PCR, we demonstrated that mdr-3 was over-expressed in both resistant lines while mdr-1 was over-expressed only in LBR-V160; in contrast, mrp-1 expression was not evidenced in any of the cell lines. After treatment with 0.1 μg ml −1 of either PSC 833 or CsA, LBR-V160 showed no changes in mdr-1 but decreased mdr-3 expression, while LBR-D160 failed to display any modification in the expression of these genes. Apoptosis was evidenced by fluorescence microscopy, S minuscule accumulation and agarose gel electrophoresis. Our results demonstrated that CsA (1 μg ml −1) was able to induce apoptosis in all cell lines: 18.31% (±4.46) for LBR-, 25.96% (±5.24) for LBR-V160 and 27.36% (±4.12) for LBR-D160, while PSC 833 (1 μg ml −1) only induced apoptosis 21.51% (±5.73) in LBR-V160 cell line. The expression of Bcl-2 family proteins (Bcl-2, Bax and Bcl-x L) was analyzed by flow cytometry showing high expression of the three proteins which was not significantly modified after treatment with either PSC 833 or CsA on the sensitive as well as on the resistant cell lines. Single stranded conformation polymorphisms analysis of p53 ( Trp53) gene in the cell lines showed no mutation in exons 5–8 of the tumor suppressor gene. We conclude that depending on the concentration used, PSC 833 and CsA may act either by modulating the mdr-3 gene (0.1 μg ml −1) or by direct impact on the cells through induction of apoptosis (1 μg ml −1), in the latter case through a mechanism that might act independent of the Bcl-2 family proteins.

Prevalent Involvement of Illegitimate V(D)J Recombination in Chromosome 9p21 Deletions in Lymphoid Leukemia

To understand molecular pathways underlying 9p21 deletions, which lead to inactivation of the p16/CDKN2A, p14/ARF, and/or p15/CDKN2B genes, in lymphoid leukemia, 30 breakpoints were cloned from 15 lymphoid leukemia cell lines. Seventeen (57%) breakpoints were mapped at five breakpoint cluster sites, BCS-LL1 to LL5, each of <15 bp. Two breakpoint cluster sites were located within the ARF and CDKN2B loci, respectively, whereas the remaining three were located >100 kb distal to the CDKN2A, ARF, and CDKN2B loci. The sequences of breakpoint junctions indicated that deletions in the 11 (73%) cell lines were mediated by illegitimate V(D)J recombination targeted at the five BCS-LL and six other sites, which contain sequences similar to recombination signal sequences for V(D)J recombination. An extrachromosomal V(D)J recombination assay indicated that BCS-LL3, at which the largest number of breakpoints (i.e. five breakpoints) was clustered, has a V(D)J recombination potential 150-fold less than the consensus recombination signal sequence. Three other BCS-LLs tested also showed V(D)J recombination potential, although it was lower than that of BCS-LL3. These results indicated that illegitimate V(D)J recombination, which was targeted at several ectopic recombination signal sequences widely distributed in 9p21, caused a large fraction of 9p21 deletions in lymphoid leukemia.

Regulation of leukemic cell adhesion, proliferation, and survival by β-catenin

AbstractIn epithelial cells β-catenin plays a critical role as a component of the cell-cell adhesion apparatus and as a coactivator of the TCF/LEF (T-cell transcription factor/lymphoid enhancer binding factor) family of transcription factors. Deregulation of β-catenin has been implicated in the malignant transformation of cells of epithelial origin. However, a function for β-catenin in hematologic malignancies has not been reported. β-Catenin is not detectable in normal peripheral blood T cells but is expressed in T–acute lymphoblastic leukemia cells and other tumor lines of hematopoietic origin and in primary lymphoid and myeloid leukemia cells. β-Catenin function was examined in Jurkat T–acute lymphoblastic leukemia cells. Overexpression of dominant-negative β-catenin or dominant-negative TCF reduced β-catenin nuclear signaling and inhibited Jurkat proliferation and clonogenicity. Similarly, these constructs inhibited proliferation of K562 and HUT-102 cells. Reduction of β-catenin expression with β-catenin antisense down-regulated adhesion of Jurkat cells in response to phytohemagglutinin. Incubation of Jurkat cells with anti-Fas induced caspase-dependent limited proteolysis of β-catenin N- and C-terminal regions and rapid redistribution of β-catenin to the detergent-insoluble cytoskeleton, concomitant with a marked decline in nuclear β-catenin signaling. Fas-mediated apoptosis was potentiated by inhibition of β-catenin nuclear signaling. The data suggest that β-catenin can play a significant role in promoting leukemic cell proliferation, adhesion, and survival.

Resveratrol inhibits the growth and induces the apoptosis of both normal and leukemic hematopoietic cells.

It is often postulated that trans-3,4',5-trihydroxystilbene (resveratrol, RES) exhibits cell growth regulatory and chemopreventive activities. However, mechanisms by which this polyphenol inhibits tumor cell growth, and its therapeutic potential are poorly understood. Using various human leukemia cells, we have first defined the anti-tumoral doses of this compound. RES inhibited the proliferation and induced the apoptosis of all tested lymphoid and myeloid leukemia cells with IC(50) = 5-43 microM. Prior to apoptosis, RES-induced caspase activity in a dose-dependent manner and cell cycle arrest in G(2)/M-phase, correlating with a significant accumulation of cyclins A and B. Leukemia cell death with RES required both caspase-dependent and -independent proteases, as it was significantly inhibited by simultaneous addition of Z-VAD-FMK and leupeptin to these cultures. While RES did not affect non-activated normal lymphocytes, this agent decreased the growth and induced the apoptosis of cycling normal human peripheral blood lymphocytes at lower concentrations (IC(50) <8 microM) than those required for most leukemia cells. RES also induced the apoptosis of early normal human CD34(+) cells and decreased the number of colonies generated by these precursor cells in a dose-dependent manner (IC(50) = 60 microM). Together, the data point to the complexity of RES-mediated signaling pathways and revealed the high anti-proliferative and proapoptotic activities of RES in normal cycling hemopoietic cells.

Decreased susceptibility of leukemic cells with PIG-Amutation to natural killer cells in vitro

AbstractThe cloning of the PIG-A gene has facilitated the unraveling of the complex pathophysiology of paroxysmal nocturnal hemoglobinuria (PNH). Of current major concern is the mechanism by which a PNH clone expands. Many reports have suggested that an immune mechanism operates to cause bone marrow failure in some patients with PNH, aplastic anemia, and myelodysplastic syndromes. Because blood cells of PNH phenotype are often found in patients with these marrow diseases, one hypothesis is that the PNH clone escapes immune attack, producing a survival advantage by immunoselection. To test this hypothesis, we examined the sensitivity of blood cells, with or withoutPIG-A mutations, to killing by natural killer (NK) cells, using 51Cr-release assay in vitro. To both peripheral blood and cultured NK cells, PIG-A mutant cells prepared from myeloid and lymphoid leukemic cell lines were less susceptible than their control counterparts (reverted from the mutant cells by transfection with a PIG-A cDNA). NK activity was completely abolished with concanamycin A and by calcium chelation, indicating that killing was perforin-dependent. There were no differences in major histocompatibility (MHC) class I expression or sensitivity to either purified perforin or to interleukin-2–activated NK cells betweenPIG-A mutant and control cells. From these results, we infer that PIG-A mutant cells lack molecules needed for NK activation or to trigger perforin-mediated killing. Our experiments suggest that PIG-A mutations confer a relative survival advantage to a PNH clone, contributing to selective expansion of these cells in the setting of marrow injury by cytotoxic lymphocytes.

Signals mediated by FcgammaRIIA suppress the growth of B-lineage acute lymphoblastic leukemia cells.

We examined Fc receptor expression and function in normal and leukemic human immature B cells. Fc receptor expression increased with normal B cell maturation: CD32(+) cells composed 8.1% +/- 1.2% (mean +/- s.d.) of the least mature (CD34(+)CD10(+)), 19.2% +/- 5.7% of intermediate (CD34(-)CD10(+)), and 82.4% +/- 5.0% of mature (CD34(-)CD10(-)) bone marrow CD19(+) B cells. Forty-five of 57 primary B-lineage acute lymphoblastic leukemia samples and all six cell lines studied expressed Fc receptors. By RT-PCR and antibody staining, FcgammaRIIA was the Fc receptor predominantly expressed in these cells. FcgammaRIIA ligation in RS4;11 and 380 cells induced tyrosine phosphorylation of CD32, CD19, CBL, SYK, P13-K p85 and SHIP, as well as RasGAP association with tyrosine-phosphorylated p62(dok). These signalling events resulted in a marked suppression of leukemia cell growth. After a 7-day exposure to anti-CD32, the recovery of ALL cells cocultured with stroma was reduced to 5.5% +/- 2.8% of control values in 380 cells (n = 14), 19.4% +/- 6.1% (n = 8) in RS4;11, and 4.0% +/- 1.3% (n = 6) in KOPN55bi. CD32 ligation also reduced cell recovery in five of seven CD32(+) primary leukemia samples. Thus, FcgammaRIIA mediates signals that suppress the growth of lymphoid leukemia cells.

High levels of chromosome aberrations correlate with impaired in vitro radiation-induced apoptosis and DNA repair in human B-chronic lymphocytic leukaemia cells

Purpose : To investigate the relationship between the susceptibility of B-chronic lymphoid leukaemia (B-CLL) cells to DNA damage-induced apoptosis, the kinetics of DNA strand-break rejoining, and chromosome damage after exposure to ionizing irradiation. Materials and methods : Lymphocytes from B-CLL patients were γ-irradiated in vitro with 0.2-5 Gy and stimulated by Staphylococcus aureus cowan I (SAC I) for estimation of chromosomal damage. Induction of apoptosis after irradiation was studied in 50 patients by two methods: morphological characterization of apoptotic cells after fluorescent staining (Hoechst), and specific quantification of mono- and oligonucleosomes in cytoplasmic cell fractions (ELISA assay). Morphological chromosome damage was scored in the first cell generation after irradiation (13 patients). In parallel, the kinetics of DNA single-strand break rejoining were investigated by the alkaline comet assay (12 patients). Results : Ionizing irradiation did not induce apoptosis in lymphocytes from a subset of B-CLL patients. The results suggest that B-CLL cells resistant to radiation-induced apoptosis could repair DNA strand-breaks more rapidly and showed a higher level of chromosome aberrations than radiation-sensitive B-CLL cells. Conclusion : Each of three biological effects observed (apoptosis, kinetics of DNA single-strand-break repair, chromosomal damage) might be explained by different modifications occurring in irradiated B-CLL cells. Their convergence strongly suggests that resistance to apoptotic death initiation by DNA damage may be impeded by a rapid engaging of the DNA repair mechanisms. The higher level of chromosome aberrations observed in these cells suggests that the type of DNA repair system involved may generate inaccurate repair.

The JAK2 inhibitor AG490 predominantly abrogates the growth of human B-precursor leukemic cells with 11q23 translocation or Philadelphia chromosome.

The Janus kinase (JAK) family is one of intracellular protein tyrosine kinases (PTKs) present in hematopoietic and lymphoid cells and has been shown to play a crucial role in a variety of biological responses. It was reported that a human B-precursor leukemic cell line was potently inhibited in its proliferation by one of synthetic PTK inhibitors (tyrphostins), AG490, via anti-JAK2 activity. However, no extensive studies about it have been performed. In the present study, we tested 16 human lymphoid leukemic cell lines (B-precursor, 12; T cell, four) for their sensitivity to AG490 using 3H-thymidine incorporation and colony formation assays, and found that B-precursor cell lines with 11q23 translocation or Philadelphia chromosome (Ph1) whose JAK2 proved to be constitutively phosphorylated were predominantly sensitive to AG490 at a concentration that has few inhibitory effect on normal hematopoiesis. We first revealed the association of JAK2 with BCR-ABL in Ph1-positive cell lines and with Bruton's tyrosine kinase (BTK) in cell lines with 11q23 translocation by coimmunoprecipitation experiments. Of interest, AG490 markedly down-regulated phosphorylation of JAK2, but rather transiently up-regulated phosphorylation of BCR-ABL and BTK, suggesting direct implication of AG490 in the process of the JAK2 dephosphorylation. These results indicate that AG490 exerts a potent inhibitory activity to B-precursor leukemia with specific chromosomal abnormalities, and a therapeutic approach using AG490 is expected.

Expression of Inducible Nitric Oxide Synthase Is Increased in Acute Myeloid Leukaemia

Significant activity of inducible nitric oxide synthase (iNOS) has been reported in tumour cells, including chronic lymphoid leukaemic cells. In this study, we analysed the expression of iNOS in 15 untreated patients with acute myeloid leukaemia (AML) and in 7 normal controls. Using flow cytometry and immunocytochemistry, we demonstrated that patients with AML had a high expression of iNOS when compared to controls. There was no correlation between the expression of iNOS and the expression of p53 and K, H, and N-ras mutation and expression, suggesting that the high expression of iNOS is independent of these proteins and could be the result of transcription factors expressed in AML.

Immunophenotypic and cytogenetic changes in acute leukaemia at relapse.

To gain more insight into the immunophenotypic and cytogenetic changes in acute leukaemia at relapse, 99 Korean patients treated at a single institution were studied: acute myelogenous leukaemia (AML, n=46), acute lymphoblastic leukaemia (ALL, n=44) and biphenotypic and mixed leukaemia (n=9). Immunophenotypic changes at relapse were observed in 51 of 99 patients (51.5%) with almost even distribution in AML and ALL. Overall expression of aberrant markers on leukaemic cells was more frequent at relapse than at initial diagnosis (P < 0.05), and this finding was most prominent in B lineage ALL (41.4% versus 10.3%, P=0.007). Gain of aberrant CD13 or CD33 at relapse of B lineage ALL was most frequently observed. Cytogenetic changes at relapse were observed in 28 of 46 patients (60.8%). The initially abnormal karyotypes were more frequently associated with clonal changes at relapse compared to initially normal karyotypes (78.3% versus 43.5%, P=0.016). Cytogenetic changes were more frequent in B lineage ALL than in AML (90% versus 47.8%, P=0.05). In ALL, patients showing cytogenetic changes at relapse were significantly younger than those showing no changes (mean age of 15.0 versus 38.8, P=0.002), whereas in AML there was no significant difference between the two groups. In conclusion, the gain of aberrant markers and cytogenetic changes at relapse, which are suggestive of clonal instability, are more prevalent in B lineage ALL compared to AML, and lymphoid leukaemic cells of younger patients are more susceptible to clonal changes at relapse.

Loss of p73 gene expression in lymphoid leukemia cell lines is associated with hypermethylation

The expression of the p73 gene and the methylation status was examined in 61 acute lymphoblastic leukemia (ALL) cell lines and lymphocytes from seven healthy individuals. p73 mRNA was not expressed in 19 (31.1%) of 61 ALL cell lines, including 11 (31.4%) of 35 B-precursor ALL cell lines, 2 (16.7%) of 12 B-ALL/Burkitt lymphoma (BL) cell lines (totally 27.7% of B-lineage cell lines), 6 (42.9%) of 14 T-ALL cell lines, and expressed in all of normal lymphocytes, by reverse transcriptase-polymerase chain reaction (RT-PCR). Restriction-enzyme related PCR (REP) and methylation-specific PCR (MSP) revealed that the cell lines lacking p73 mRNA expression were hypermethylated. In contrast, normal lymphocytes and most cell lines that expressed detectable p73 mRNA were not hypermethylated with the exception of five cell lines. Furthermore, bisulfite genomic sequencing confirmed the results obtained by REP and MSP. Our results suggest that p73 inactivation may be involved in the pathogenesis of both T- and B-ALLs, and that hypermethylation is the predominant mechanism of inactivation of the p73 gene in ALL.

Effect of granulocyte colony-stimulating factor on chemotherapeutic activity of cytosine arabinoside in acute leukemic cell lines.

Recent studies have shown the presence of receptors for granulocyte colony-stimulating factor (G-CSF) on lymphoid leukemic cells. To determine the effect of G-CSF on chemotherapeutic activity of cytosine arabinoside (Ara-C) on lymphoid as well as myeloid leukemic cells, we evaluated cell counts, apoptosis, and growth inhibition in HL-60, KG-1, Molt-4, Jijoye, and CCRF-CEM cell lines after incubation with Ara-C (0.1 and 1 micromol/L) and/or 5 ng/mL G-CSE G-CSF potentiated the effect of Ara-C on 2 of 3 lymphoid leukemic cell lines (Molt-4 and Jijoye), whereas it decreased the apoptosis and the effect of Ara-C on myeloid cell lines (HL-60 and KG-1).

Improved Dissolution and Cytotoxicity of Camptothecin Incorporated into Oxidized-Cellulose Microspheres Prepared by Spray Drying

Oxidized celluloses (OC) containing 7, 13, and 20% carboxylic content (OC-7, OC-13, and OC-20, respectively) have been converted into aqueous colloidal dispersions and used to prepare microspheres of the antineoplastic agent camptothecin (CPT) by spray drying. Plasticizers used were glycerin, polyethylene glycol 400 (PEG-400), and polyethylene glycol 6000 (PEG-6000). Irrespective of the carboxylic content of OC and the nature of plasticizer employed, the size of microspheres varied from 1.25 ± 0.40 to 1.52 ± 0.47 μm. The release studies in pH 7.4 buffer revealed the dissolution of CPT to be faster from the microsphere formulations than from physical mixtures and free CPT. The times to release 50% CPT (T-50%) from microspheres prepared using OC-7, OC-13, and OC-20 were about 31, 37, and 19 h, respectively. The in vitro cytotoxicity results indicated OC-20/CPT microspheres to be more effective than free CPT against human-derived RPMI-8402 lymphoid and THP-1 myeloid leukemia cell lines. The ED50 values for the OC-20/CPT microspheres and free CPT were 1 × 10−5 and 0.25 × 10−1 μg/mL, respectively, against the RPMI-8402 line and 0.5 × 10−2 and 0.75 μg/mL, respectively, against the THP-1 line. The higher activity of OC-20/CPT microspheres compared to that of the free drug is attributed to increased dissolution of CPT from microspheres.

2-(Acyloxy)ethylphosphonate analogues of prenyl pyrophosphates: synthesis and biological characterization

2-(Acyloxy)ethylphosphonate analogues of geranyl, farnesyl, and geranylgeranyl pyrophosphate have been prepared. Horner–Wadsworth–Emmons condensation of different terpene aldehydes with an unsymmetrical bisphosphonate was the key step in syntheses of the phosphonates bearing α,β-unsaturated acyloxy groups. After preparation of the respective phosphonic acids through reaction with TMSBr, both acids and esters were tested for their effects on DNA synthesis in human-derived myeloid and lymphoid leukemia cell lines. The phosphonate esters varied substantially in their ability to impair proliferation of the different cell lines, but testing against one possible target, farnesyl protein transferase (FPTase), revealed little impact at concentrations ranging up to 10 μM. Because the corresponding 2,3-dihydro compounds showed similar biological activity, conjugate addition would not appear to be involved in the toxicity.

Expression of thrombopoietin receptor and its functional role in human B-precursor leukemia cells with 11q23 translocation or Philadelphia chromosome

Thrombopoietin (TPO) is a hematopoietic growth factor which plays a central role in normal megakaryocytopoiesis and thrombopoiesis. Although the interaction between TPO and its receptor c-Mpl encoded by the c-mpl gene is now known to be implicated in the proliferation and/or differentiation of abnormal myeloid cells and normal hematopoietic stem cells, little is known about a role of the TPO/c-Mpl system in lymphoid leukemia cells. In the present study, we first examined the expression of c-mpl/c-Mpl in 23 human lymphoid leukemic cell lines (T-lineage 4, B-lineage 19) using three distinct methods. The c-mpl mRNA was detectable in as many as 20 cell lines (T-lineage 3, B-lineage 17) by reverse transcriptase-polymerase chain reaction, but its translated product, c-Mpl, was demonstrable by Western blot only in B-lineage cell lines. Flow cytometric analysis revealed the surface c-Mpl expression in 13 of 17 B-lineage cell lines, but its higher expression (>40%) was restricted in nine B-precursor cell lines, eight of which had 11q23 translocation or Philadelphia chromosome (Ph1). We also demonstrated that two of eight cell lines with 11q23 translocation or Ph1 exhibited a significant proliferative response to TPO in the 3H-thymidine uptake and colony-forming assays. Triggering of these cell lines by TPO transiently up-regulated tyrosine phosphorylation of JAK-2 and Shc, indicating that their receptor is functional. Primary leukemia cells separated from patients with B-precursor acute lymphoblastic leukemia with Ph1 or 11q23 translocation also showed the surface c-Mpl expression and a significant responsiveness to TPO. These results suggest that the TPO/c-Mpl interaction may play a physiological role in the growth regulation of B-precursor leukemia cells particularly with specific chromosomal abnormalities.

Intracellular triggering of Fas, independently of FasL, as a new mechanism of antitumor ether lipid-induced apoptosis.

Antitumor ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3); edelfosine) induces apoptosis in cancer cells, sparing normal cells. We have found that the apoptotic action of ET-18-OCH(3) required drug uptake and Fas in the target cell. Failure to accomplish one of these requirements prevents cell killing by the ether lipid. In human lymphoid leukemic cells, ET-18-OCH(3) does not promote Fas or FasL expression and ET-18-OCH(3)-induced apoptosis is not inhibited by pre-incubation with an anti-Fas blocking antibody that abrogates cell killing mediated by Fas/FasL interactions. ET-18-OCH(3)-resistant normal human Fas-positive fibroblasts do not incorporate ET-18-OCH(3), but undergo apoptosis upon ET-18-OCH(3) microinjection. Murine fibroblasts L929 and L929-Fas, stably transfected with human Fas cDNA, do not incorporate ET-18-OCH(3) and are resistant to its action when added exogenously. Microinjection of ET-18-OCH(3) induces apoptosis in L929-Fas cells, but not in wild-type L929 cells. Confocal laser scanning microscopy shows that ET-18-OCH(3) induces Fas clustering and capping during triggering of ET-18-OCH(3)-induced apoptosis. Microinjection-induced apoptosis and Fas clustering are specific for the molecular structure of ET-18-OCH(3). Our data indicate that ET-18-OCH(3) induces apoptosis via Fas after the ether lipid is inside the cell, and this Fas activation is independent of the interaction of Fas with its natural ligand FasL. This explains the selective action of ET-18-OCH(3) on tumors since only cancer cells incorporate sufficient amounts of the drug.

Cellular adaptation to down-regulated iron transport into lymphoid leukaemic cells: effects on the expression of the gene for ribonucleotide reductase

Ribonucleotide reductase is an iron-containing enzyme that is essential for DNA synthesis. Whereas previous studies have used various iron chelators to examine the relationship between cellular iron metabolism and ribonucleotide reductase activity in cells, they have not elucidated the relationship between iron transport into cells and the expression of the gene for ribonucleotide reductase. To investigate this, we examined ribonucleotide reductase mRNA, protein and enzyme activity in a novel line of CCRF-CEM cells (DFe-T cells) that display an approx. 60% decrease in their uptake of iron compared with the parental wild-type cell line. We found that DFe-T cells displayed an approx. 40% decrease in ribonucleotide reductase specific enzyme activity relative to wild-type cells without a change in their proliferation. Kinetic analysis of CDP reductase activity revealed an approx. 60% decrease in Vmax in DFe-T cells without a change in Km. Despite the decrease in enzyme activity, the mRNA and protein for the R1 and R2 subunits of ribonucleotide reductase in DFe-T cells were similar to those of wild-type cells. ESR spectroscopy studies revealed that DFe-T cells had a 22% decrease in the tyrosyl free radical of the R2 subunit, suggesting that a larger amount of R2 protein was present as functionally inactive apo-R2 in these cells. Our studies indicate that ribonucleotide reductase activity in CCRF-CEM cells can be down-regulated by more than 50% in response to down-regulated iron transport without an adverse effect on cell proliferation. Furthermore, our studies suggest a regulatory link between ribonucleotide reductase activity and iron transport into these cells.