Resistance to nitric oxide-mediated apoptosis in HL-60 variant cells is associated with increased activities of Cu,Zn-superoxide dismutase and catalase

Nitric oxide (NO) released from (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (DETA/NO or NOC-18) induces apoptosis in human leukemia HL-60 cells. In this study, we isolated a HL-60 variant cell line, HL-NR6, that is resistant to DETA/NO toxicity as assessed by DNA fragmentation, morphology, and colony forming ability. The variant cells also showed resistance to reactive oxygen species (ROS) such as superoxide and hydrogen peroxide as well as NO donors, but not to anti-tumor drugs. We found that HL-NR6 cells when compared with HL-60 cells possessed twice the activities of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and catalase, but no change in Mn-SOD nor in glutathione peroxidase. Immunoblotting confirmed the high levels of both enzymes in the variant cell. We also observed that ROS generation following DETA/NO exposure was substantially higher in HL-60 cells than in HL-NR6 cells, using the 2',7'-dichlorofluorescein fluorometric method. Moreover, the SOD mimetic Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin and exogenous catalase effectively attenuated DETA/NO-elicited DNA fragmentation in HL-60 cells. Taken together, these data suggested that the NO resistance in HL-NR6 cells is associated with the increased Cu,Zn-SOD/catalase and that NO-mediated apoptosis in HL-60 cells is correlated with the generation of ROS and derived molecules like peroxynitrite.     

A lineage-specific protein kinase crucial for myeloid maturation

To identify genes involved in macrophage development, we used the differential display technique and compared the gene expression profiles for human myeloid HL-60 leukemia cell lines susceptible and resistant to macrophage maturation. We identified a gene coding for a protein kinase, protein kinase X (PRKX), which was expressed in the maturation-susceptible, but not in the resistant, cell line. The expression of the PRKX gene was found to be induced during monocyte, macrophage, and granulocyte maturation of HL-60 cells. We also studied the expression of the PRKX gene in 12 different human tissues and transformed cell lines and found that, among these tissues and cell types, the PRKX gene is expressed only in blood. Among the blood cell lineages, the PRKX gene is specifically expressed in macrophages and granulocytes. Antisense inhibition of PRKX expression blocked terminal development in both the leukemic HL-60 cells and normal peripheral blood monocytes, implying that PRKX is a key mediator of macrophage and granulocyte maturation. Using the HL-60 cell variant deficient in protein kinase C-beta (PKC-beta) and several stable PKC-beta transfectants, we found that PRKX gene expression is under control of PKC-beta; hence PRKX is likely to act downstream of this PKC isozyme in the same signal transduction pathway leading to macrophage maturation.     

A secreted proform of neutrophil proteinase 3 regulates the proliferation of granulopoietic progenitor cells

Myeloid leukemia cells, the human promyelocytic cell line HL-60, and a subpopulation of normal marrow cells produce a leukemia-associated inhibitor (LAI) that reversibly downmodulates DNA synthesis of normal granulopoietic progenitor cells colony-forming unit granulocyte-macrophage (CFU-GM). We isolated an active 125-kD component of LAI from HL-60 conditioned medium (CM), subjected it to cyanogen bromide cleavage and show by amino acid sequencing of the resulting peptides that it consists of a complex of the serine proteinase inhibitor alpha1-antitrypsin and a 31-kD fragment that retained the S-phase inhibitory activity, but resisted sequencing. This finding suggested that the 31-kD fragment originated from one of the neutrophil serine proteases (ie, elastase, proteinase 3, or cathepsin G) produced by normal promyelocytes, as well as HL-60 cells, for storage in primary granules and partly secreted during synthesis as enzymatically inactive proforms. Immunoblot analysis showed that the 125-kD complex contained proteinase 3 (PR3), and immunoprecipitation of PR3 from HL-60 CM abrogated the S-phase inhibitory activity, whereas immunoprecipitation of cathepsin G or elastase did not. Immunoprecipitation of PR3 from CM of a subpopulation of normal marrow cells also abrogated the S-phase inhibitory effect. Furthermore, CM from rat RBL and murine 32D cell lines transfected with human PR3 both reduced the fraction of CFU-GM in S-phase with 30% to 80% at 1 to 35 ng/mL PR3, whereas CM of the same cells transfected with cathepsin G or elastase did not. Also, an enzymatically silent mutant of PR3 exerted full activity, showing that the S-phase modulatory effect is not dependent on proteolytic activity. Amino acid sequencing of biosynthetically radiolabeled PR3 showed that PR3 from transfected cells is secreted after synthesis as proforms retaining amino terminal propeptides. In contrast, mature PR3 extracted from mature neutrophils has only minor activity. The inhibitory effect of secreted PR3 is reversible and abrogated by granulocyte (G)- or granulocyte-macrophage colony-stimulating factor (GM-CSF). Experiments with highly purified CD34(+) bone marrow cells suggested that PR3 acts directly on the granulopoietic progenitor cells. These observations suggest a role for PR3 in regulation of granulopoiesis, and possibly in suppression of normal granulopoiesis in leukemia.     

Transport and activation of the vacuolar aspartic proteinase phytepsin in barley (Hordeum vulgare L.)

The primary translation product of barley aspartic proteinase, phytepsin (EC 3.4.23.40), consists of a signal sequence, a propart, and mature enzyme forms. Here, we describe post-translational processing and activation of phytepsin during its transport to the vacuole in roots, as detected by using metabolic labeling and immunoprecipitation. After removal of the signal sequence, the glycosylated precursor of 53 kDa (P53) was produced and further processed to polypeptides of 31 and 15 kDa (P31 + P15) and, subsequently, to polypeptides of 26 and 9 kDa (P26 + P9), 45 min and 24 h after synthesis, respectively. The processing occurred in a late-Golgi compartment or post-Golgi compartment, because brefeldin A inhibited the processing, and P53 acquired partial endoglycosidase H resistance 30 min after synthesis, whereas P15 was completely resistant. The N-glycosylation inhibitor tunicamycin had no effect on transport, but the absence of glycans on P53 accelerated the proteolytic processing. Phytepsin was also expressed in baculovirus-infected insect cells. The recombinant prophytepsin underwent autoproteolytic activation in vitro and showed enzymatic properties similar to the enzyme purified from grains. However, a comparison of the in vitro/in vivo processing sites revealed slight differences, indicating that additional proteases are needed for the completion of the maturation in vivo.     

Ethical concerns in modern medical practice

Differentiation of HL-60 cells along the granulocytic lineage by DMSO in the presence of transforming growth factor-beta and low concentrations of 1,25-dihydroxyvitamin D3 leads to the upregulation of 5-lipoxygenase activity in 100,000 g supernatants and intact cells to levels which are comparable to normal granulocytes. Similarly, differentiation of the human monocytic cell line Mono Mac 6 by 1,25-dihydroxyvitamin D3 and transforming growth factor-beta strongly upregulates the 5-lipoxygenase pathway. Here, we describe an assay system for leukotriene biosynthesis inhibitors which is based on the in-vitro differentiation of HL-60 and Mono Mac 6 cells. Different leukotriene biosynthesis inhibitors like the nonredox type inhibitor ZM 230487, the redox type inhibitor BW A4C and the FLAP inhibitor MK886 were tested and the results were compared with an assay system based on normal human granulocytes. ZM 230487, BWA4C and MK886 showed similar potencies in these cell lines as compared to normal leukocytes. Thus, the in-vitro differentiation of HL-60 and Mono Mac 6 cells provides an excellent model for the screening of drugs affecting the 5-lipoxygenase pathway.     

Fibronectin-mediated cell adhesion is required for induction of 92-kDa type IV collagenase/gelatinase (MMP-9) gene expression during macrophage differentiation. The signaling role of protein kinase C-beta

Induction of the 92-kDa gelatinase (MMP-9) gene expression is associated with macrophage differentiation. In this study, we explored the regulatory mechanisms underlying this differentiation-associated MMP-9 gene expression in human HL-60 myeloid leukemia cells and human peripheral blood monocytes. Phorbol 12-myristate 13-acetate (PMA) markedly induced MMP-9 gene expression in HL-60 cells; the induction closely paralleled the timing and extent of PMA-induced cell adhesion and spreading, a hallmark of macrophage differentiation. Similarly, treatment with PMA or macrophage-colony stimulating factor stimulated adherence and spreading of blood monocytes with a concurrent 7- or 5-fold increase in MMP-9 production, respectively. In protein kinase C (PKC)-beta-deficient HL-60 variant cells (HL-525), PMA failed to induce cell adhesion and MMP-9 gene expression. Transfecting HL-525 cells with a PKC-beta expression plasmid restored PKC-beta levels and PMA inducibility of cell adhesion and spreading as well as MMP-9 gene expression. Induction of cell adhesion and MMP-9 gene expression in HL-60 cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to fibronectin (FN) and its receptor alpha5 beta1 integrin. HL-525 cells, which constitutively display high levels of surface alpha5 beta1 integrin, adhered and spread on immobilized FN with concomitant induction of MMP-9 gene expression. Cytochalasins B and D were each a potent inhibitor of MMP-9 production. Our results suggest that alpha5 beta1 integrin-mediated interaction of immature hematopoietic cells with FN plays a critical role in modulating matrix-degrading activities during macrophage differentiation.     

Regulation and function of Bcl-2 during differentiation-induced cell death in HL-60 promyelocytic cells

Polymorphonuclear leukocytes are generated by differentiation of early myeloid precursors. Once fully differentiated, blood neutrophils are programmed to die rapidly and are removed by tissue macrophages. In normal myeloid cells, the death mechanism seems to be coupled to the differentiation pathway and is accomplished by a process termed apoptosis. In the present study, we have examined the role of Bcl-2 in the differentiation pathways of the promyelocytic cell line HL-60. Treatment of HL-60 with retinoic acid or phorbol ester, which induced neutrophil or macrophage-like cell differentiation, respectively, resulted in progressive loss of cellular viability and internucleosomal DNA degradation. In HL-60, differentiation and apoptosis were coupled to down-regulation of the Bcl-2 protein. Overexpression of Bcl-2 by gene transfer inhibited apoptosis triggered by terminal differentiation of HL-60. Yet, Bcl-2 did not alter the expression of surface markers or other phenotypic changes that are induced upon myeloid differentiation. In contrast to HL-60, another immature myeloid cell line, K562, did not produce Bcl-2 but expressed a related protein, Bcl-xL, that functions as a repressor of apoptotic cell death. K562 has been shown to be relatively resistant to a variety of apoptotic stimuli. Incubation of HL-60 and K562 with inhibitors of macromolecular synthesis induced apoptosis, which appeared earlier in HL-60 than in K562. Interestingly, Bcl-2 overexpression protected K562 cells from apoptosis induced by inhibitor of macromolecular synthesis but it had little or no effect on HL-60 cells. We conclude that although differentiation and apoptosis proceed simultaneously, they can be uncoupled by expression of Bcl-2. Down-regulation of Bcl-2 appears to be part of the differentiation pathway and may serve to facilitate the apoptotic response.     

Paget''s disease of bone

The protein encoded by ras and src protooncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study, we investigated the effect of oncogenic p21ras and Py-MT/pp60c-src on the synthesis of syndecan-1, a membrane anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control. To this end, we used Caco-2 cells transfected with an activated (Val-12) human Ha-ras gene or the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. As compared to control vector-transfected Caco-2 cells, both oncogene-transfected cells exhibited: (1) a decrease in syndecan-1 specific activity; (2) a decrease in size and sulfation of syndecan-1 ectodomain glycosaminoglycan side chains; and (3) an active heparanase specifically degrading the heparan sulfate chains. In conclusion, the tumorigenic progression induced by oncogenic p21ras or Py-MT/pp60c-src is associated with marked alterations of syndecan-1 at the post-translational level.     

Elimination of human leukemia by monoclonal antibodies in an athymic nude mouse leukemia model

A human acute myeloid leukemia model has been developed by i.v. transplantation of HL-60 myeloid leukemia cells into Swiss nude mice pretreated with cyclophosphamide. HL-60 cells disseminated into hematopoietic tissues as determined by flow cytometric analysis, fluorescence microscopy, fluorescence in situ hybridization analysis, and colony formation assay. Passive immunotherapy using murine anti-CD13 (F23) or anti-CD33 (M195) mAbs was able to eliminate completely the HL-60 cells in the mice, as determined by fluorescence in situ hybridization analysis, colony formation assay, and culture of mouse blood and tissue cells in vitro. Although F23 is able to inhibit completely CD13/aminopeptidase N enzymatic activity, actinonin, another potent inhibitor of CD13/aminopeptidase N, was not active as an antileukemic agent. HL-60 cell surface antigens, including CD13 (aminopeptidase N) and CD33 (p67), down-regulated over time, and murine anti-HL-60 antibody was generated while the cells grew in the mice. This response was suppressed by cyclophosphamide. These data suggest that leukemia cell elimination was antibody mediated.     

Apolipoprotein B is intracellularly associated with an ER-60 protease homologue in HepG2 cells

Two ALLN (N-acetyl-leucyl-leucyl-norleucinal)-sensitive endoplasmic reticulum (ER)-localized proteases (ER-60 and ER-72) were recently purified from rat liver. We used an antibody to rat ER-60 to investigate the possible role of this protease in apolipoprotein B (apoB) degradation. First, immunoprecipitation and immunoblotting experiments with the anti-rat ER-60 antibody suggested that HepG2 cells contain a homologue of ER-60 with an approximate molecular mass of 58-60 kDa. The ER-60 homologue was mostly associated with the luminal contents of HepG2 microsomes. Evidence from co-immunoprecipitation and cross-linking experiments appear to suggest that the ER-60 homologue in HepG2 cells is associated with apoB intracellularly. A small pool of apoB was recovered when HepG2 lysates were subjected to immunoprecipitation with anti-rat ER-60 antibody followed by a second immunoprecipitation with anti-apoB antibody. Furthermore, cross-linking of permeabilized cells with dithiobis(succinimidylpropionate) further demonstrated association of apoB with the ER-60 homologue in HepG2 cells. Three polypeptides with molecular masses of 78, 66, and 50 kDa were consistently found to be associated with apoB as well as the 58-kDa ER-60 homologue. The 78-kDa protein associated with both apoB and ER-60 appeared to represent immunoglobulin heavy chain-binding protein (BiP) based on immunoprecipitation with a monoclonal antibody. Cross-linking and immunoblotting experiments suggested the association of the 78-kDa BiP with both the 58-kDa ER-60 homologue as well as the 550-kDa apoB. In summary, the data suggests that HepG2 cells contain a 58-kDa protein which is homologous to the rat liver ER-60 in size, antigenecity, and intracellular localization. The ER-60 homologue in HepG2 cells appears to be closely associated with apoB, as well as other proteins possibly representing ER chaperones such as BiP. We hypothesize that the ER-60 homologue may be involved in the degradation of apoB in the ER lumen of HepG2 cells.     

Cellular pharmacology of mitoxantrone in p-glycoprotein-positive and -negative human myeloid leukemic cell lines

Previous reports suggest that resistance to mitoxantrone in different tumor cell lines is unrelated to the overexpression of p-glycoprotein. In order to determine the role of p-glycoprotein in the cellular pharmacology of mitoxantrone flow cytometry and confocal microscopy were used to study two human myeloid leukemia cell lines selected for resistance to mitoxantrone (HL-60MX2) and doxorubicin (HL-60DOX). To optimize the detection of intracellular mitoxantrone, we determined the maximum excitation (607 nm) and emission (684 nm) wavelength by fluorescence spectroscopy. The modified flow cytometric conditions using 568.2 nm laser emission for excitation and a 620 nm long pass filter for fluorescence collection resulted in a 1-log increase in sensitivity, compared with standard 488-nm laser excitation. Uptake and retention of mitoxantrone in the presence of verapamil, a calcium channel blocker known to inhibit p-glycoprotein, were analyzed. Our results showed no change in uptake and retention of the drug in p-glycoprotein-negative mitoxantrone-resistant HL-60MX2 cells and in its sensitive parental line, HL-60s. In contrast, 3.1- and 2.4-fold increases were found in uptake and retention of mitoxantrone in p-glycoprotein-positive cells (HL-60DOX) incubated with verapamil. Confocal microscopy of intracellular drug distribution demonstrated reduced nuclear uptake, which could be reversed by verapamil, in HL-60DOX. A characteristic punctate pattern was observed for the intracytoplasmic drug distribution in HL-60DOX and HL-60MX2 cells and was partially modified by the presence of verapamil in HL-60DOX cells. Verapamil increased cytotoxicity of mitoxantrone two-fold in HL-60DOX cells, 1.4-fold in HL-60MX2, and had no effect in HL-60s. Our study demonstrates that the cellular pharmacology of mitoxantrone is affected by p-glycoprotein and can be reversed at least in part by verapamil. Other mechanisms of resistance however, seem to play a determinant role in the modulation of mitoxantrone cytotoxicity.     

Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein

Rap1 small GTP-binding protein has the same amino acid sequence at its effector domain as that of Ras. Rap1 has been shown to antagonize the Ras functions, such as the Ras-induced transformation of NIH 3T3 cells and the Ras-induced activation of the c-Raf-1 protein kinase-dependent mitogen-activated protein (MAP) kinase cascade in Rat-1 cells, whereas we have shown that Rap1 as well as Ras stimulates DNA synthesis in Swiss 3T3 cells. We have established a cell-free assay system in which Ras activates bovine brain B-Raf protein kinase. Here we have used this assay system and examined the effect of Rap1 on the B-Raf activity to phosphorylate recombinant MAP kinase kinase (MEK). Recombinant Rap1B stimulated the activity of B-Raf, which was partially purified from bovine brain and immunoprecipitated by an anti-B-Raf antibody. The GTP-bound form was active, but the GDP-bound form was inactive. The fully post-translationally lipid-modified form was active, but the unmodified form was nearly inactive. The maximum B-Raf activity stimulated by Rap1B was nearly the same as that stimulated by Ki-Ras. Rap1B enhanced the Ki-Ras-stimulated B-Raf activity in an additive manner. These results indicate that not only Ras but also Rap1 is involved in the activation of the B-Raf-dependent MAP kinase cascade.     

A- and B-type lamins are differentially expressed in normal human tissues

A selection of normal human tissues was investigated for the presence of lamins B1, B2, and A-type lamins, using a panel of antibodies specific for the individual lamin subtypes. By use of immunoprecipitation and two-dimensional immunoblotting techniques we demonstrated that these antibodies do not cross-react with other lamin subtypes and that a range of different phosphorylation isoforms is recognized by each antibody. The lamin B2 antibodies appeared to decorate the nuclear lamina in all tissues examined, except hepatocytes, in which very little lamin B2 expression was observed. In contrast to previous studies, which suggested the ubiquitous expression of lamin B1 in mammalian tissues, we show that lamin B1 is not as universally distributed throughout normal human tissues as was to be expected from previous studies. Muscle and connective tissues are negative, while in epithelial cells lamin B1 seemed to be preferentially detected in proliferating cells. These results correspond well with those obtained for lamin B1 in chicken tissues. The expression of A-type lamins is most prominent in well-differentiated epithelial cells. Relatively undifferentiated and proliferating cells in epithelia showed a clearly reduced expression of A-type lamins. Furthermore, most cells of neuroendocrine origin as well as most hematopoietic cells were negative for A-type lamin antibodies.     

Effect of a vitamin D3 analog, EB1089, on hematopoietic stem cells from normal and myeloid leukemic blasts

The seco-steroid 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induces differentiation and inhibits clonal proliferation of HL-60 cells. We analyzed the effect of a novel vitamin D3 analog, EB1089, on normal myeloid and leukemic cells as well as CD34+ cells. EB1089 showed an extraordinary inhibition of clonal growth of HL-60 cells (ED50 = 5 x 10(-11) M) and AML blast cells (ED50 = 9 x 10(-10) M) compared to 1,25(OH)2D3 without suppression of growth of normal human bone marrow CFU-GM. The CD34+ cells from acute myeloid leukemia (AML) blasts were inhibited in a dose-dependent fashion by 1,25(OH)2D3 with an ED50 of 1.2 x 10(-9) M; and even more strikingly, 10(-10) M of EB1089 inhibited all clonal growth of human CD34+ leukemic colony-forming cells. In contrast, both EB1089 and 1,25(OH)2D3 (10(-8) M) showed little or only mild inhibition of CD34+ clongenic hematopoietic cells from normal human peripheral blood (PB); and in liquid culture, EB1089 stimulated the proliferation of normal human CD34+ cells about 2.5 times as compared to control cultures. In order to evaluate the potential use of EB1089 for purging leukemic cells from normal CD34+ progenitor cells for PB stem cell transplantation (PBSCT), normal human PB mononuclear cells (PBMNC) were contaminated with HL-60 cells, and then CD34+ cells purified and treated with EB1089. We found that CD34+ purification and EB1089 purging was able to eliminate approximately 100% of HL-60 leukemic cells with no toxicity to normal CD34+ hematopoietic progenitor cells. These data suggested that purification of CD34+ cells and ex vivo treatment with EB1089 might provide an effective therapeutic approach for PBSCT.     

Viral determinants of HIV-1 sufficient to extend tropism to macrophages are distinct from the determinants that control the cytopathic phenotype in HL-60 cells

DESIGN: Infection of the human promyelocytic cell line HL-60 with NL4-3, a molecularly cloned HIV-1 strain that productively infects T cells, results in adaptation of the virus and production of a variant, NL4-3(M). Unlike NL4-3, NL4-3(M) has a rapid cytopathic effect in HL-60 and other myeloid cell lines. OBJECTIVE: To demonstrate that the tropism of NL4-3(M) is extended to primary monocyte-derived macrophages (MDM), and to determine whether the envelope gene, env, of NL4-3(M) is responsible for cytopathicity in HL-60 cells and replication in MDM. METHODS: A chimeric virus (NL4-3envA) containing the majority of env of NL4-3(M) was generated, and tested for virus replication and cytopathic effect in H9 and HL-60 cells, as well as for virus replication in primary MDM. To assess virus replication, the cultures were analyzed for expression of viral envelope glycoproteins on the infected cells and production of extracellular HIV-1 p24 antigen. Cytopathic effect on HL-60 cells was evaluated by monitoring the viabilities of the cultures. In addition, the majority of env of NL4-3envA was sequenced. RESULTS: The biological phenotypes of NL4-3, NL4-3(M), and NL4-3envA are distinctly different. Although both NL4-3(M) and NL4-3envA replicate in MDM, only NL4-3(M) is rapidly cytopathic in HL-60 cells. Nine amino-acid changes were identified within the envelope glycoproteins of NL4-3envA compared with NL4-3. CONCLUSIONS: The viral determinants of NL4-3(M) sufficient to extend the tropism of this virus to MDM reside, in part, in env. These genetic determinants are distinct from the viral determinants that control the cytopathic phenotype of this virus in HL-60 cells.     

Mitogenic and oncogenic properties of the small G protein Rap1b

It has been widely reported that the small GTP-binding protein Rap1 has an anti-Ras and anti-mitogenic activity. Thus, it is generally accepted that a normal physiological role of Rap1 proteins is to antagonize Ras mitogenic signals, presumably by forming nonproductive complexes with proteins that are typically effectors or modulators of Ras. Rap1 is activated by signals that raise intracellular levels of cAMP, a molecule that has long been known to exert both inhibitory and stimulatory effects on cell growth. We have now tested the intriguing hypothesis that Rap1 could have mitogenic effects in systems in which cAMP stimulates cell proliferation. The result of experiments addressing this possibility revealed that Rap1 has full oncogenic potential. Expression of Rap1 in these cells results in a decreased doubling time, an increased saturation density, and an unusual anchorage-dependent morphological transformation. Most significantly, however, Rap1-expressing cells formed tumors when injected into nude mice. Thus, we propose that the view that holds Rap1 as an antimitogenic protein should be restricted and conclude that Rap1 is a conditional oncoprotein.     

Spinal tuberculosis in an unusual location

OBJECTIVE: To study the effect of transforming growth factor beta 1 on tumor cells. METHOD: HL-60 cells were transfected with transforming growth factor beta 1 expression vector by electroporation. Stable cell clones resistant to G 418 were obtained after 2-3 week growth with the drug-containing medium. A series of malignant phenotype were compared between transfected and untransfected HL 60 cells. RESULTS: The growth speed of HL-60 cells which overexpressed transforming growth factor beta 1 mRNA decreased significantly. Clonigenecity in soft agarase and tumor formation in athymic mice were lower than those untransfected HL-60 cells. CONCLUSION: Suppression effect of transforming growth factor beta 1 on malignant phenotype in HL-60 cells were demonstrated.