Lymphoid Leukemia Cells Research Articles

Association between Lyn protein tyrosine kinase (p53/56lyn) and the beta subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in a GM-CSF-dependent human megakaryocytic leukemia cell line (M-07e).

The role of the lyn product (p53/p56lyn), a membrane-associated protein tyrosine kinase in the signaling pathway used by granulocyte macrophage-CSFR (GM-CSFR) was investigated by using the GM-CSF-dependent human megakaryoblastic leukemia cell line M-07e. M-07e cells express GM-CSFR and are dependent on GM-CSF for survival and proliferation in vitro. Treatment with anti-lyn Abs coimmunoprecipitated, along with lyn product, the beta subunit of GM-CSFR and a phosphoprotein with a molecular mass of 120 kDa (p120) in the lysates of M-07e cells but not in the lysates of human monocyte-derived macrophages (HMDM) or human lymphoid leukemia cells. That the 120-kDa phosphoprotein coimmunoprecipitated by anti-lyn Abs is the beta subunit of GM-CSFR was confirmed in the immunoprecipitates (IP) of M-07e cells with the use of an agarose-conjugated anti-p-tyr mAb. The formation of GM-CSF/GM-CSFR/lyn signaling complexes was verified in an autoradiographic study with anti-lyn IP of M-07e cells that had been bound with 125I-labeled recombinant human (rh)GM-CSF. The p120 protein (beta subunit) was not detected in the IP of M-07e cells with anti-fyn or anti-PI3 Abs. A direct association of Lyn kinase with the beta subunit of GM-CSFR was illustrated with a reversed approach showing the recovery of Lyn protein in anti-beta (CRS1) but not anti-alpha IP of M-07e cells that had been starved for a prolonged period. Finally, the interaction of Lyn kinase with the GM-CSFR complexes was further corroborated using anti-GM-CSF (G133) mAb, which coimmunoprecipitated both the p120 beta subunit and lyn product in the lysates of M-07e cells that had been bound with rhGM-CSF before cell lysis. Removal of rhGM-CSF from culture medium for 10 to 12 h resulted in a marked decrease in lyn-associated kinase activity but not the beta subunit/lyn kinase complex formation. Taken together, our results showed that, in M-07e cells, Lyn protein tyrosine kinase (p53/p56lyn) is stably associated with a constitutively phosphorylated beta subunit of the GM-CSFR in a manner that seems to be independent of lyn kinase activity.

Enhancement of cytogenetic damage and of antineoplastic effect in lymphoid L1210 leukemia cells treated with prostaglandin E2 and cyclophosphamide in vivo

An enhanced frequency of sister-chromatid exchanges (SCEs) and increased cell division delays induced by cyclophosphamide (CP) were observed when lymphoid L1210 leukemia cells were post-treated in vivo with prostaglandin E2 (PGE2). CP gave a slight, non-significant increase in survival while PGE2 gave a slight, non-significant decrease in survival. However, PGE2 in combination with CP was found to have a non-significant potentiating effect on survival in comparison with mice treated with CP alone. In mice treated with the combined CP (5 μg/g b.w.) plus PGE2 (2 μg/g b.w.) treatment, a significant ( P<0.01) enhancement of survival time in comparison with the untreated controls was observed.

Expression of c-kit receptor (CD117) and CD34 in leukemic cells.

The expression of c-kit receptor (c-kit R; CD117) and CD34 was examined in acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML) in blastic transformation (BT), and myelofibrosis (MF) in myeloid BT. In myeloid leukemia including AML, CML-myeloid BT and MF-myeloid BT, both c-kit R and CD34 were expressed synchronously, while in lymphoid leukemia including ALL and CML-lymphoid BT, only CD34 was highly expressed. A close correlation between c-kit R and CD33 expression and an inverse correlation between c-kit R and CD19 expression were observed when all of the myeloid plus lymphoid leukemia cells were analysed. There was a close correlation between c-kit R and CD34 expression in the myeloid leukemia cells. c-kit R expression may be associated with myeloid phenotypes of leukemic cells and may be useful for the diagnosis of myeloid leukemia. The literature of c-kit R expression in leukemic cells is reviewed here and the comparison of c-kit R and CD34 expression in normal hematopoietic progenitor cells with those on the leukemic counterparts was discussed.

Hypolipidemic, anti-obesity, anti-inflammatory, anti-osteoporotic, and anti-neoplastic properties of amine carboxyboranes.

The amine-carboxyborane derivatives were shown to be effective antineoplastic/cytotoxic agents with selective activity against single-cell and solid tumors derived from murine and human leukemias, lymphomas, sarcomas, and carcinomas. The agents inhibited DNA and RNA synthesis in preference to protein synthesis in L1210 lymphoid leukemia cells. Inosine-monophosphate dehydrogenase apparently is a target site of the compounds; similar effects on phosphoribosyl-pyrophosphate amido transferase, orotidine-monophosphate decarboxylase, and both nucleoside and nucleotide kinases were observed. Deoxyribonucleotide pool levels were reduced in the cells; DNA strand scission was observed with the agents. In rodents, the amine carboxyboranes were potent hypolipidemic agents, lowering both serum cholesterol and triglyceride concentrations, in addition to lowering cholesterol content of very low-density lipoprotein and low-density lipoprotein (LDL) and elevating high-density lipoprotein (HDL) cholesterol concentrations. De novo regulatory enzymes involved in lipid synthesis were also inhibited (e.g., hypocholesterolemic 3-hydroxy-3-methyl-Coenzyme A reductase, acyl-Coenzyme A cholesterol acyltransferase, and sn-glycerol-3-phosphate acyltransferase). Concurrently, the agents modulated LDL and HDL receptor binding, internalization, and degradation, so that less cholesterol was delivered to the plaques and more broken down from esters and conducted to the liver for biliary excretion. Tissue lipids in the aorta wall of the rat were reduced and fewer atherosclerotic morphologic lesions were present in quail aortas after treatment with the agents. Cholesterol resorption from the rat intestine was reduced in the presence of drug. Genetic hyperlipidemic mice demonstrated the same types of reduction after treatment with the agents. The agents would effectively lower lipids in tissue based on the inhibition of regulatory enzymes in pigs. These findings should help improve domestic meat supplies from fowl and pigs. The amine-carboxyboranes were effective anti-inflammatory agents against septic shock, induced edema, pleurisy, and chronic arthritis at 2.5 to 8 mg/kg. Lysosomal and proteolytic enzyme activities were also inhibited. More significantly, the agents were dual inhibitors of prostaglandin cyclooxygenase and 5'-lipoxygenase activities. These compounds also affected cytokine release and white cell migration. Subsequent studies showed that the amine-carboxyboranes were potent anti-osteoporotic agents reducing calcium resorption as well as increasing calcium and proline incorporation into mouse pup calvaria and rat UMR-106 collagen.

Induction of cell surface interleukin 2 receptor alpha chain expression on non-T lymphoid leukemia cells

We examined nine cases of adult non-T lymphoid leukemia to investigate the cell surface inducibility of interleukin 2 receptor α chain (IL-2Rα) and β chain (IL-2Rβ) after in vitro culture with and without recombinant human interleukin-1β (rhIL-1β). Induction of IL-2Rα was observed in four of six cases with precursor B-cell acute lymphoblastic leukemia (pre-B ALL) and in all of three cases with B-cell mature lymphoid neoplasm (two chronic lymphocytic leukemia and one leukemic phase of non-Hodgkin's lymphoma). All of the IL-2Rα-inducible cases could express this spontaneously even without rhIL-1β, while IL-2Rβ did not appear on leukemic cells from any of the cases tested. IL-2Rα-inducible pre-B ALL cases displayed stem cell antigen CD34 and induced myeloid-associated antigen CD13 simultaneously. These results suggest that IL-2Rα but not IL-2Rβ is easily inducible in certain cases of mature B-cell lymphoid neoplasm and pre-B ALL with immature characteristics.

Cytotoxic Evaluation of Some 3,5-Diarylidene-4-piperidones and Various Related Quaternary Ammonium Compounds and Analogs

A number of 3,5-diarylidene-4-piperidones (1) and some related quaternary ammonium salts (5) as well as closely related analogs were prepared principally as candidate cytotoxic agents in two screens. The first test system used an average of 54 human tumor cell lines from eight neoplastic diseases, namely leukemia, melanoma, colon, non-small-cell lung, small-cell lung, central nervous system, ovarian, and renal cancers. Selective toxicity was demonstrated by some of the compounds, especially toward leukemia. The second screen used L 1210 lymphoid leukemia cells. In general, the compounds were less cytotoxic than the reference drug melphalan in both screens. Linear plots were made between the Hammett (σ), fragment (f), and molar refractivity (MR) constants of the nuclear substituents in series1and5with the IC50figures of both the human tumor cell lines and L 1210 cells. Evaluation against the human tumor cell lines revealed that increases in thefvalues were correlated with elevation of cytotoxicity in both series1and5; MR constants were also important in series5. In the L 1210 screen, σ and MR constants were positively correlated with cytotoxicity. X-ray crystallography was undertaken on 3,5-bis-[[4′-(methylthio)phenyl]methylene]-1-methyl-4-piperidone methiodide (5d), which had significant cytotoxicity, and 3,5-bis(4-pyridylmethylene)-1-methyl-4-piperidone methiodide (6), which was virtually inactive in both screens. A number of structural features were noted, and in particular a smaller interplanar angle made by one of the arylidene groups with the central piperidine ring in the case of5dthan either of the 4-pyridylmethylene moieties in6may contribute to the variation in cytotoxicity between these two compounds. Eleven compounds which were examined for activity against a panel of 12 viruses were shown to display only marginal potencies or to be inactive at the highest concentrations utilized. Two compounds with slight activity both had the hydrophilic carboxylate ion in the aryl rings. From this study, various conclusions pertaining to future molecular modification with a view to increasing cytotoxic potency were made.

Synthesis and Cytotoxic Evaluation of Some Cyclic Arylidene Ketones and Related Oximes, Oxime Esters, and Analogs

A number of arylidene derivatives of alicyclic ketones and some corresponding oximes, oxime esters, and related compounds were prepared as candidate cytotoxic agents. All of the compounds were evaluated against murine L1210 lymphoid leukemia cells. In general, cytotoxicity was greatest with the α,β‐unsaturated ketones and diminished with the oximes, and the oxime esters had little or no activity in this screen. When the same compounds were examined in both thein vitroL1210 and P388 leukemia screens, in the majority of cases the L1210 cells were more sensitive to these derivatives. Over half of the compounds prepared were evaluated against approximately 55 human tumorsin vitroand showed selective toxicity toward one or more groups of neoplastic diseases, particularly leukemia. Some correlations between structure and bioactivity were discerned. The cytotoxicity screening and stability studies of representative compounds suggested that the ketones, oximes, and oxime esters were stable under the conditions of bioevaluation. X‐ray crystallography of four representative compounds revealed structural features associated with cytotoxicity which may be considered in the design of future candidate cytotoxins.

Transduction of the IL2 gene into human acute leukemia cells: induction of tumor rejection without modifying cell proliferation and IL2 receptor expression.

Previous studies have suggested that some of the limitations associated with the administration of high-dose exogenous interleukin 2 (IL2) may be overcome, at least partly, by cytokine gene transfer modalities. These findings have prompted investigations into whether human tumor cells may be transduced with the IL2 gene and whether tumor cell lines could be engineered to release IL2. The purpose of this study was to evaluate the possibility of inducing a productive transfer of the IL2 gene into human acute leukemia cells and to assess the phenotypic and proliferative changes generated in the engineered cells, as well as their tumorigenic potential in nude mice. Three retroviral vectors (DC/TK/IL2, DC/AD/R/IL2, and N2/CMV/IL2) carrying the IL2 gene were used to transduce three human leukemic cell lines: K562 and U937 (myeloid) and ST4 (lymphoid). Messenger RNA expression of the IL2 gene was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and productive IL2 release using a human IL2 assay and an enzyme-linked immunosorbent assay kit. The expression of the p55 (alpha) and p75 (beta) chains of the IL2 receptor were determined by RT-PCR and indirect immunofluorescence. The kinetics of in vitro growth and proliferation of parental and engineered cells were also measured. Parental and IL2 gene-transduced ST4 lymphoblasts were injected into immunosuppressed nude mice that had their tumors measured twice weekly. The productive insertion of the IL2 gene was achieved in all three cell lines studied. The amounts of IL2 constitutively released by the engineered neoplastic cells ranged between 1 and 11 U/mL of IL2 produced from 10(6) cells in 72 hours. A fivefold increase in IL2 production was obtained in ST4 cells by further limiting dilution cloning of the bulk-infected cells. The stable integration of the IL2 gene did not modify the phenotype of the leukemic cells, the expression of the IL2 receptor alpha and beta chains and of several cytokine genes, or the kinetics of in vitro growth and proliferation. In nude mice injected with various IL2-producing ST4 clones, tumor growth associated inversely with the amounts of IL2 secreted by the leukemic cells. The results of this study demonstrate that the IL2 gene can be productively transduced into human myeloid and lymphoid leukemic cells without modifying their phenotypic and proliferative properties and that this transduction leads to a reduced or abrogated in vivo tumorigenic potential.

Interleukin 1 (IL-1α and IL-1β) induces differentiation/activation of B cell chronic lymphoid leukemia cells

The effect of interleukin 1 (IL-1) was studied on leukemic cells from 12 patients with B cell chronic lymphoid leukemias including two cases of hairy cell leukemia (HCL), two cases of HCL-variant (HCL-V), one case of prolymphocytic leukemia (PLL), and seven cases of chronic lymphocytic leukemia (CLL). In most cases, IL-1 induced differentiation characterized by increments of sIgγ +, sIgμ + and PCA-1 + cells, and a decrement of CD5 + cells, and activation characterized by increments of CD23 + and HC2 + cells, but induced the proliferation of leukemic cells only in two HCL-V cases. Among these effects, increment of sIg + cells was observed more frequently in non-CLL ( 4 5 cases) than in CLL ( 2 7 cases), and increments of sIgγ +, CD23 + and PCA-1 + cells were induced more frequently by IL-1β than by IL-1α. These results suggest that IL-1, especially IL-1β, plays a significant role in the differentiation and activation of leukemic cells, but has only a minor role as an extrinsic/autocrine growth factor. IL-1 may be useful for studying the differentiation pathway of leukemic cells in B cell chronic lymphoid leukemias, particularly those with more mature cells such as PLL and HCL.

C-kit expression in human megakaryoblastic leukemia cell lines

A panel of 164 continuous human leukemia-lymphoma cell lines was analyzed for expression of c-kit using Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). The c-kit transcripts were detectable in cell lines assigned to the myeloid (in 7 of 29 by Northern blotting and in 4 of 8 by RT-PCR), monocytic (in 1 of 24 by Northern blotting and in 3 of 6 by RT-PCR), erythroid (in 6 of 8 by Northern blotting and in 5 of 5 by RT-PCR), and megakaryoblastic (in 10 of 10 by Northern blotting) lineages, c-kit expression was not seen by Northern blotting or RT-PCR analysis in any of the 93 lymphoid leukemia, myeloma, or lymphoma cell lines. Treatment of four megakaryoblastic cell lines with protein kinase C activators (phorbol ester 12-O-tetradecanoylphorbol 13-acetate and Bryostatin 1) led to terminal differentiation as assessed by morphologic alterations, changes in the surface marker profile, and growth arrest. These effects were associated with enhanced c-kit mRNA expression. Exposure to all- trans retinoic acid down-regulated c-kit mRNA levels, while simultaneously causing morphologic alterations in all four cell lines. Stimulation with growth factors (interleukin-3, granulocyte macrophage- colony stimulating factor, and insulin-like growth factors I and II), used to assess any role of c-kit in proliferative processes, did not lead to significant upregulation or downregulation of c-kit expression. The finding of constitutive and high expression of c-kit mRNA in all megakaryoblastic leukemia cell lines and its modulation by various reagents might further contribute to the understanding of megakaryopoietic proliferation, differentiation, and leukemogenesis.

C-kit Expression in Human Megakaryoblastic Leukemia Cell Lines

AbstractA panel of 164 continuous human leukemia-lymphoma cell lines was analyzed for expression of c-kit using Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). The c-kit transcripts were detectable in cell lines assigned to the myeloid (in 7 of 29 by Northern blotting and in 4 of 8 by RT-PCR), monocytic (in 1 of 24 by Northern blotting and in 3 of 6 by RT-PCR), erythroid (in 6 of 8 by Northern blotting and in 5 of 5 by RT-PCR), and megakaryoblastic (in 10 of 10 by Northern blotting) lineages, c-kit expression was not seen by Northern blotting or RT-PCR analysis in any of the 93 lymphoid leukemia, myeloma, or lymphoma cell lines. Treatment of four megakaryoblastic cell lines with protein kinase C activators (phorbol ester 12-O-tetradecanoylphorbol 13-acetate and Bryostatin 1) led to terminal differentiation as assessed by morphologic alterations, changes in the surface marker profile, and growth arrest. These effects were associated with enhanced c-kit mRNA expression. Exposure to all-trans retinoic acid down-regulated c-kit mRNA levels, while simultaneously causing morphologic alterations in all four cell lines. Stimulation with growth factors (interleukin-3, granulocyte macrophage-colony stimulating factor, and insulin-like growth factors I and II), used to assess any role of c-kit in proliferative processes, did not lead to significant upregulation or downregu-lation of c-kit expression. The finding of constitutive and high expression of c-kit mRNA in all megakaryoblastic leukemia cell lines and its modulation by various reagents might further contribute to the understanding of megakaryopoietic proliferation, differentiation, and leukemogenesis.

Changes in c-myc expression and the kinetics of dexamethasone-induced programmed cell death (apoptosis) in human lymphoid leukaemia cells.

The kinetics of dexamethasone-induced death of CCRF CEM clone C7A human lymphoblastic leukaemia cells was determined with respect to changes in the expression of the c-myc protein. Cell death was characterised as being by apoptosis: cells with an intact plasma membrane had condensed chromatin and were characterised as having approximately 300 kbp fragments when DNA integrity was analysed by pulsed-field electrophoresis. Onset of apoptosis required a minimum of 36 h exposure to 5 microM dexamethasone; before this time no apoptotic cells were observed. This 36 h incubation period appeared to be necessary to prime the cells for subsequent death by apoptosis. In the continued presence of dexamethasone the percentage of apoptotic cells increased to 60% apoptotic cells by 54 h. Investigation of changes in c-myc protein showed that it was undetectable after 12 h of incubation with dexamethasone, although cells were not committed to die at this time. Cells were treated with dexamethasone for 54 h and for various pulsed periods with a non-toxic concentration of cycloheximide (200 nM). When cycloheximide was present during the first 36 h priming period of dexamethasone treatment, there was an immediate loss of c-myc protein and apoptosis at 54 h was completely inhibited. In contrast, there was no inhibition of apoptosis when dexamethasone-treated cells were incubated with an 18 h pulse of cycloheximide added after 36 h. Cells exposed to dexamethasone for 36 h ('primed') were given various periods of dexamethasone-free incubation before readdition of dexamethasone for a further 18 h. The longer the cells were free of drug after priming, the less susceptible they became to apoptosis, suggesting a slow decay of their 'memory' of the initial 36 h period of exposure. Cycloheximide inhibited the decay of this memory. Removal of dexamethasone after a 36 h exposure was characterised by a subsequent 24 h suppression of c-myc protein expression. Despite this, 90% of cells became refractory to apoptosis before the reappearance of c-myc protein. The evidence does not support the hypothesis that changes in c-myc expression are required for the engagement of apoptosis of CEM cells.

Effects of herbimycin A and its derivatives on growth and differentiation of Ph 1-positive acute lymphoid leukemia cell lines

The molecular basis of the Philadelphia chromosome (Ph 1) is a structurally altered c-abl ( bcr/abl) gene which encodes an abnormally large protein with protein tyrosine kinase activity. Herbimycin A, an inhibitor of tyrosine kinase, preferentially inhibited the growth of Ph 1-positive acute lymphoid leukemia (ALL) cell lines, as well as Ph 1-positive chronic myeloid leukemia (CML) cell lines. Although noncytotoxic concentrations of herbimycin A induced erythroid differentiation of two CML-derived cell lines, K562 and KU812, in a previous study, the differentiation-inducing effect of herbimycin A on Ph 1-positive ALL cell lines was less strong. Herbimycin A enhanced some differentiation-associated properties of one Ph 1-positive ALL cell line, L2, but the effect of herbimycin A on the other Ph 1-positive ALL cell lines was cytotoxic rather than cytostatic (differentiation-inducing). Several derivatives of herbimycin A were synthesized and their effects on the cell proliferation of Ph 1-positive CML and ALL cell lines were examined. The sensitivities of the Ph 1-positive cell lines to herbimycin A derivatives were different from the data on the rat kidney cell line infected with Rous sarcoma virus (v- src) derived from a previous study, suggesting bcr/abl kinase may differ in sensitivity from other tyrosine kinases. Moreover, the sensitivities of the ALL cell lines were not the same as those of the CML cell lines. These results suggest that a specific inhibitor of bcr/abl kinase could be an effective antileukemic agent against Ph 1-positive CML or ALL.

Foundations In Cancer Research Hemopoietic Regulators And Leukemia Development: A Personal Retrospective

Publisher Summary This chapter presents a personalized analysis of the work done in the last 40 years on the possible involvement of specific hemopoietic regulators in the development of leukemia. The studies on the thymus focus on the puzzling autonomous behavior of the organ. The data indicates that the enormous proliferation of T lymphocytes occurring within the organ was essentially being regulated locally by thymic stroma1 and epithelial cells. The experiments leading to the accidental discovery of clonal hemopoietic cultures were carried for a quite different purpose. Attempts were made to culture murine lymphoid leukemic cells. The work, over the last 40 years, began with studies on lymphoid leukemia but evolved through the circumstances to be concerned mainly with myeloid leukemia. The four colony-stimulating factors are characterized and identified as the major regulators of granulocyte-macrophage populations.

Adhesion molecule expression on B-cells from acute and chronic lymphoid leukemias.

Adhesion molecule expression on acute and chronic lymphoid leukemia cells of B lineage (B-ALL and B-CLL) may subserve several functions. Adhesion of leukemic cells to endothelial cells and to extracellular matrix components is relevant to homing, trafficking and spread of the malignant cells, and thus to clinical presentation, course and disease prognosis. Adhesive interactions between malignant cells and accessory cells, particularly stromal cells in the bone marrow environment, may support growth of the malignant cells via cytokine-delivered messages. They may also deliver signals that prevent or trigger programmed cell death of tumor cells. Here we review data on the adhesive phenotype of leukemic blasts from pro-B (CALLA +) ALL and of cells from B-CLL cases. We show that expression of certain adhesion molecules may help define disease subsets with distinctive clinical and prognostic features. One adhesion molecule, the lymphocyte homing receptor CD44, allows definition of two groups of B-CLL patients with significantly different survival.

Expression and modulation of annexin VIII in human leukemia-lymphoma cell lines

Annexin VIII is a calcium- and phospholipid-binding protein with anticoagulant activity. Annexin VIII mRNA was found to be specifically expressed in acute promyelocytic leukemia (APL) cells; it was not found in other types of acute myeloid leukemia (AML) nor in lymphoid malignancies. Using Northern blot analysis we investigated annexin VIII expression in 142 continuous human leukemia and lymphoma cell lines at the mRNA level. While the only APL cell line, NB-4, was indeed positive, other cell lines also displayed annexin VIII mRNA: 4 22 myeloid cell lines, 8 23 monocytic cell lines, 2 8 megakaryoblastic cell lines, 5 26 lymphoma-derived cell lines, 2 10 myeloma cell lines and 1 44 lymphoid leukemia cell lines. The strongest expression was seen in NB-4 and in the Hodgkin's disease derived cell line HDLM-2. Treatment of NB-4 cells with all- trans retinoic acid (ATRA) or the phorbol ester TPA induced terminal differentiation and down-regulated annexin VIII mRNA expression rapidly within a few hours; vitamin D3 was ineffective in this regard; the protein kinase C activator Bryostatin 1 up-regulated the expression. A panel of initially negative cell lines could not be induced by any of these biomodulators to transcribe annexin VIII. The half-life ( T 1 2 ) of annexin VIII mRNA was about 3–4 h using actinomycin D as transcription inhibitor. Treatment with ATRA or TPA prior to exposure to actinomycin shortened the T 1 2 to 2 h while Bryostatin 1 extended it to 6 h. As 21 141 non-APL cell lines were positive, annexin VIII cannot be used as a marker gene for APL cells; however, it might be associated with myelomonocytic or erythro-megakaryoblastic precursor cells. Annexin VIII gene expression might play a unique role in the proliferation and/or differentiation of leukemic cells and could be associated with the particular abnormal hemostasis of some leukemias.

Structure-dependent antitumor activities of novel anthracyclines YM1, YM3, YM4 and YM6: drug transport properties and effects on biomacromolecule synthesis in drug sensitive and resistant leukemia cells.

The antitumor activities of four novel doxorubicin (DOX) analogues, YM1, YM3, YM4 and YM6 in relation to their structure and drug transport properties, have been investigated in U937 monocytic and CCRF-CEM lymphoid drug sensitive leukemia cell lines, as well as in CEM/VLB100, a drug resistant subline displaying high levels of P-glycoprotein. Treatment of all cell lines with YM1, 3, 4 and 6 produced a dose-dependent decrease in DNA, RNA and protein synthesis as measured by [3H]-thymidine, [3H]-uridine and [3H]-leucine uptake respectively. YM1 was more effective than YM3, YM4 or YM6 against the drug sensitive cells. The antitumor effects of all these DOX-analogues on macromolecule synthesis in U937 and CCRF-CEM cells were lower than that of DOX and epirubicin (EDR). A rapid accumulation of the novel anthracyclines was found in all cell lines compared with DOX or EDR. However, the maximal accumulation of the DOX-analogues was lower than that of EDR. There is a greater efflux from CCRF-CEM sensitive cells and less from CEM/VLB100 resistant cells of the DOX-derivatives when compared with EDR and DOX. Drug-induced cytotoxicity significantly correlated (P < 0.05) with drug retention levels in CCRF-CEM and U937 drug sensitive cells as indicated by an inverse correlation curve between anthracycline retention and drug-induced IC50 value. It was demonstrated that an increased level of drug retained within the sensitive cells would therefore produce a more cytotoxic effect of the drug. However, no such correlation was observed in CEM/VLB100 resistant cells. YM3 was shown to have an increased antitumor activity against CEM/VLB100 resistant cells compared with DOX with a lower resistance factor. These results showed that the antitumor effects of four novel DOX-analogues, like DOX or EDR, were associated with inhibition of DNA replication, transcription and translation. The finding that resistant leukemic cells are more susceptible to the cytotoxic effect of YM3 than DOX warrants further investigation to identify the intrinsic mechanism of resistance.

The effects of boron containing peptides on L1210 lymphoid leukemia metabolism

The purpose of this study was to establish the efficacy and mode of action of peptide boron derivatives as antineoplastic agents and to evaluate their safety in vivo. Boron-containing phenylalanine and tyrosine methyl esters were found to be potent cytotoxic agents in a number of murine and human cancer cell lines. DNA, RNA and protein syntheses were inhibited by selected agents, e.g. [(trimethylamine boryl)carbonyl]-phenylalanine-acetyl ester (9) andN-acetyl-p-boron-phenyl-alanyl-phenlalanine-methyl ester (10), in L1210 lymphoid leukemia cells. IMP dehydrogenase, OMP decarboxylase, m-RNA, t-RNA, r-RNA polymerase and ribonucleoside reductase activities were inhibited. d(CTP) levels were reduced. DNA strand scission occurred after 24 hr incubation. Acute toxicity studies in mice demonstrated that the key derivative was safe at therapeutic levels with no effects on histology of major organs, hematopoietic parameters and clinical values.

Modulation of transferrin receptor mRNA by transferrin-gallium in human myeloid HL60 and lymphoid CCRF-CEM leukaemic cells.

Gallium binds to the iron transport protein transferrin (Tf), is incorporated into cells through transferrin receptors (TfR) and inhibits iron-dependent DNA synthesis. Since cellular TfR expression is tightly regulated by the availability of iron, we investigated the effects of transferrin-gallium (Tf-Ga) on TfR mRNA levels in myeloid HL60 and lymphoid CCRF-CEM cells. In HL60 cells, Tf-Ga increased TfR mRNA levels in a dose-dependent fashion. This increase in TfR mRNA was blocked by Tf-Fe and by cycloheximide. Analysis of the rate of mRNA decay in the presence of actinomycin D revealed that the half-life of TfR mRNA was increased in HL60 cells incubated with Tf-Ga. The rate of transcription of TfR mRNA was not increased by Tf-Ga. In contrast with HL60 cells, CCRF-CEM cells displayed a decrease in the level of TfR mRNA after incubation with Tf-Ga. Tf-Ga inhibited iron uptake in both HL60 and CCRF-CEM cells but increased the level of TfR mRNA only in HL60 cells, suggesting that the Tf-Ga induction of TfR mRNA was not solely due to inhibition of cellular iron uptake. At growth-inhibitory concentrations, Tf-Ga increased the TfR mRNA level in HL60 cells but decreased it in CCRF-CEM cells. Our studies suggest that in HL60 cells, gallium regulates TfR expression at the post-transcriptional level by mechanisms which require de novo protein synthesis and involve interaction with iron. The divergent effects of Tf-Ga on TfR mRNA in myeloid HL60 and lymphoid CCRF-CEM cells suggest that differences exist in the regulation of TfR expression between these two cell types.

Myeloperoxidase: Expression and Modulation in a Large Panel of Human Leukemia-Lymphoma Cell Lines

AbstractMyeloperoxidase (MPO) is found exclusively in the azurophilic granules (primary lysosomes) of normal myelomono-cytic cells. Cytochemical staining for MPO activity is used clinically to distinguish myeloid from lymphoid leukemias. We studied the expression of MPO at the RNA and protein level in 140 continuous human leukemia-lymphoma cell lines using classical cytochemistry, immunofluorescent staining with a specific monoclonal antibody, Northern blot analysis, and a reverse transcription-polymerase chain reaction (RT-PCR) amplification assay. Seventy-eight lymphoid leukemia, myeloma, and lymphoma cell lines were negative; only 3 pre-B-acute lymphoblastic leukemia (ALL) cell lines were MPO-positive. Two of these MPO-po-sitive pre-B-ALL cell lines showed a trace expression after RT-PCR and Southern blotting corresponding to 4% to 6% of the transcripts found in other positive myeloid cell lines. The third pre-B-ALL cell line was positive in Northern blots and cytochemical/immunofluorescent staining; however, only few cells were weakly positive in the latter assay. Although 15 of 59 cell lines assigned to the myeloid, monocytic, megakaryocytic, or erythroid lineages were MPO-positive in Northern blots, those 15 and 13 additional cell lines showed bands of mRNA after RT-PCR. MPO protein was detected in all 16 Northern-positive cell lines; on the other hand, there were 4 cell lines that were protein-positive, but Northern-negative. Differentiation induced by protein kinase C activators 12-O-tetradecanoylphorbol 13-acetate and Bryostatin 1 or by all-trans retinoic acid was associated with a decrease in MPO mRNA in all 7 initially positive cell lines studied, even leading to the complete absence of transcripts, but the enzymatic activity of the differentiated cells was only slightly less than that of unstimulated cells. MPO expression could not be induced in 10 initially negative cell lines. The half-life of MPO mRNA was found to be about 6 hours and was not shortened by prior exposure of the cells to the differentiation-inducing agents. These results confirm that MPO expression is mainly associated with myelomonocytic cells, but also underline the notion that MPO cannot be used as an absolutely lineage-specific marker for the distinction of leukemic cells. MPO can be used as an excellent parameter to characterize the various stages of normal and induced differentiation.