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Studies are described employing two erythropoietic systems to elucidate regulatory mechanisms that control both normal erythropoiesis and erythroid differentiation of transformed hemopoietic precursors. Evidence is provided suggesting that normal erythroid cell precursors require erythropoietin as a growth factor that regulates the number of precursors capable of differentiating. Murine erythroleukemia cells proliferate without need of erythropoietin; they show a variable, generally low, rate of spontaneous differentiation and a brisk rate of erythropoiesis in response to a variety of chemical agents. Present studies suggest that these chemical inducers initiate a series of events including cell surface related changes, alterations in cell cycle kinetics, and modifications of chromatin and DNA structure which result in the irreversible commitment of these leukemia cells to erythroid differentiation and the synthesis of red-cell-specific products.

Transport of fluorescent derivatives of fatty acids across the cell membrane of cultured human leukemic myeloid cells (HL 60) and their subsequent metabolic utilization were studied. The rates of uptake of these derivatives and their incorporation into cellular lipids wer compared with that of radioactively labelled palmitic acid. Three groups of fluorescent derivatives were observed: A, those transported into the cells and subsequently incorporated into neutral lipids and phospholipids, B, fatty acids which were taken up by the cells but not utilized metabolically, and C, fatty acids which were not transported across the cell membrane. Fatty acids of the latter group, except the hydrophobic probe, also contained functional groups such as hydroxy, acetylamino or sulfonylamino. When observed in fluorescence microscopy, cells incubated with group A fatty acids contained intracellular fluorescent granules, whereas those incubated with group B fatty acids showed diffuse fluorescence. HL 60 cells undergo differentiation into granulocytes or macrophages upon treatment with dimethylsulfoxide or a phorbol ester, respectively. When compared to the uninduced cells, the transport of the fluorescent fatty acids or palmitic acid as well as their subsequent incorporation into lipids were considerably lower in the granulocytes and higher in the macrophages. The use of the fluorescent derivatives as a tool for studying transport of fatty acids across the cell membrane is discussed.

Introduction of DNA into human hemopoietic cells is required for the study of regulatory mechanisms operating in these cells, as well as for possible procedures of gene therapy. However, with hemopoietic cells the conventional technique of calcium phosphate precipitation is inefficient. The pathway of encapsidation of plasmid DNA as simian virus 40 (SV40) pseudovirions for the introduction of new genetic material was therefore investigated. Encapsidation was achieved in COS (monkey kidney) cells, which express SV40 large tumor (T) antigen constitutively. The vector, pSO, was introduced to the COS cells by DNA transfection. It carried the SV40 origin of replication (ori), to facilitate replication of the plasmid in the COS cells. The SV40 capsid proteins were supplied in trans by a helper SV40 virus. The bacterial chloramphenicol acetyltransferase gene cat was used as a model for gene transmission. After encapsidation, the pseudovirions were used in infection of the human erythroleukemic cell line K562 and of normal human bone marrow cells. The results demonstrate that the cat gene can be transmitted with high efficiency. Over 40% of the infected K562 cells and 30% of the infected bone marrow cells were observed to contain plasmid DNA 48 hr after infection. Moreover, the results suggest that the efficiency of gene transmission by this vector can be improved and so may approach the theoretical 100%.

Previous studies demonstrated that 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a tumor promoter, is a potent inhibitor of inducer-mediated differentiation of murine erythroleukemia cells. Inhibition of cell differentiation was associated with inhibition of cell growth. The present studies, employing a cell line adapted for growth in TPA, demonstrate that inhibition of differentiation is not dependent upon inhibition of cell growth or a change in the cell division cycle; neither is inhibition of differentiation accompanied by detectable effect on cell uptake of [3H]hexamethylene bisacetamide, the inducer used in these studies. TPA causes an inhibition of expression of all hexamethylene bisacetamide-inducible erythroid characteristics measured, including commitment to terminal cell division, accumulation of globin mRNA, and synthesis of globins, spectrin, heme synthetic enzymes (delta-aminolevulinic acid dehydratase and uroporphyrinogen-I synthase) and heme. A hypothetical model for the inhibitory action of tumor promoters on terminal cell differentiation is discussed.

Addition of the potent tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), to murine erythroleukemia cell lines in suspension cultures inhibited both spontaneous differentiation and differentiation induced by hexamethylene bisacetamide (HMBA), dimethyl sulfoxide, or butyric acid. Inhibition was unrelated to cytotoxicity and was reversible. When several plant diterpenes were tested, there was a positive correlation between tumor-promoting activity and inhibition of differentiation. TPA inhibited HMBA-induced differentiation only if added prior to the time of commitment to differentiation, as assayed by scoring for differentiation after transfer of cells from HMBA to fresh medium without HMBA. TPA-mediated inhibition of differentiation was associated with a decrease in globin mRNA accumulation.

Proteolytic activity was measured in human leukemic promyelocytic cell line (HL-60) grown in culture, before and after the addition of agents which promote differentiation. The 36000 X g soluble fraction of the cells degraded [14C]globin with maximal activity at pH 3.6, while the insoluble fraction had a pH optimum at 8.0. This pattern did not change upon differentiation. The acid protease activity of the soluble fraction increased following differentiation. After 4 days in the presence of dimethyl sulfoxide, the differentiated cells exhibited 4-fold higher specific activity as compared with 4 day-old control cells. In contrast, the alkaline activity of the insoluble fraction of the differentiated cells was 4-fold lower than that of the undifferentiated cells. It is suggested that the changes in enzyme activities may serve the new functions acquired by the mature granulocytes.

A new myelomonoblastic cell line (M20) was established from the peripheral blood of a ten-year-old child with acute myeloblastic leukemia, using an improved method for supporting the initial stages of cell proliferation. The addition of irradiated macrophage monolayers to the proliferating cells appeared to overcome the deterioration of the primary cultures and enable them to continue proliferating until they became independent of this environment. The cell line that developed consisted of myeloblasts and promyelocytes characterized by light and scanning electron microscopy, cytochemistry, and enzymatic activities. The cells expressed Fc receptors and WT1 antigens but did not exhibit HLA-DR, HMA1, Epstein-Barr virus nuclear antigen, and surface Ig. The M20 cells produced colonies when cultured in semisolid medium and secreted lysozyme, prostaglandin E2, and interleukin 1. An attempt was also made to analyse the position of the M20 cells in the scheme of differentiation of the myelomonocytic lineage using different approaches. Treatment of the cells with 12-O-tetradecanoyl phorbol 13-acetate induced their adherence to plastic surfaces and partial maturation to macrophages as judged by morphological criteria, cytochemistry, and enzyme activities. However, comparison of the M20 cells to other well-established myelomonoblastic cell lines did not reveal any pattern suggesting a possible relationship between surface markers, cell function, and differentiation pathway of the various cell lines tested. Establishment of additional cell lines and identification of new markers may assist in defining the mechanisms involved in normal differentiation and malignant transformation of this cell lineage. In addition, such cell lines may also provide a tool for the quantitative recovery of a variety of monokines.

Human myeloid leukemia cells can be induced to differentiate into macrophage-like cells by various phorbol esters, particularly 12-O-tetradecanoyl-phorbol-14-acetate (TPA). In this study, the effect of several known protease inhibitors on TPA-induced differentiation of human acute promyelocytic leukemia cells (line HL-60) was tested. Among the test compounds, only pentamidine-isethionate (PI), an inhibitor of trypsin-like enzymes, prevented one early marker of differentiation, e.g. cell adherence to plastic and glass surfaces. However, PI failed to affect other markers of differentiation and did not inhibit readherence of scraped and resuspended TPA-treated cells. Exposure to TPA resulted in a decrease in the cellular alkaline proteolytic activity and an increase in the acid proteolytic activity. PI further inhibited the residual activity of the alkaline protease in the 36,000 g pellet fraction of the TPA-treated cells, but did not reduce this activity in control cells. The present results indicate, on the basis of the differential effects of PI, that the emergence of differentiation markers in HL-60 cells following exposure to TPA is independent of the induction of adherence.

Incubation of cells with 12-(1-pyrene) dodecanoic acid (P12) followed by irradiation with ultraviolet light at 366 nm (UVA) resulted in cytotoxicity. We compared the photosensitivity to UVA irradiation of various human myelo-monocytic leukemic cell lines, their intra- and inter-clonal variability and correlated their photosensitivity to P12-uptake and metabolism. The fluorescence properties of pyrene were utilized for flow cytometric analysis of cell distribution with respect to P12-uptake as well as for sorting subpopulations differing in their fluorescence. Spectrofluorometric analysis of the total cell-associated fluorescence and of the cellular lipids-associated fluorescence were also carried out. Considerable heterogeneity in P12-uptake and photosensitivity was found not only among cell lines, but also in the response of different clones and among the individual cells in specific clonal populations. Within a clone, photosensitivity was related to the amount of P12 taken up by the individual cells, while among different cell lines and their clones the photosensitivity was correlated with the proportion of cellular pyrene-linked phospholipids. The larger the fraction of pyrene-linked phospholipids within the cell--the more sensitive it was to UVA-irradiation. Photosensitivity could be affected by changing the proportion of cellular pyrene-linked phospholipids. Cells treated with cAMP showed an increase in total P12-uptake, but the proportion of pyrene-linked phospholipids was reduced, resulting in lower photosensitivity. These findings, demonstrating that by manipulating lipid metabolism photosensitivity can be modified, may prove useful in a clinical setting for selective photosensitization of malignant cells.

The presence of myelomonocytic progenitor cells in human peripheral blood was used for the analysis of cloned populations of human monocytes. Colonies of granulocytes and macrophages were obtained by plating peripheral blood mononuclear cells (PBM) in methylcellulose containing medium in the presence of medium conditioned by nonstimulated PBM (CM). Following 20-25 days of incubation, most colonies were found to consist of cells with monocyte-macrophage morphology. Cloned populations of monocytes were tested for several monocyte membrane markers and compared to noncloned adherent monocytes. HLA-DR, 63D3, LeuM2 antigens and Fc receptors were expressed on cells from individual colonies in similar proportions to their expression on noncloned monocytes. Some colonies were uniform in their negative expression of the 63D3 antigen, as were the noncloned monocytes. Although the clonality of cells tested was not directly proven, these results indicated that at least for some monocyte markers, heterogeneous expression was obtained in monoclonal populations of monocytes. It is possible, however, that testing of additional markers and functions may reveal homogeneous clones of monocytes and suggest the existence of stable subsets.

Murine erythroleukemia (MEL) and human K-562 cell lines were cultured in the presence of 57Fe, and the quantities of cellular iron-containing compounds were determined with the aid of Mössbauer spectroscopy. Upon induction of differentiation, both ferritin-iron and hemoglobin (Hb) iron could be detected. Treatment of the cells with 0.01%-0.02% acetylphenylhydrazine (APH) resulted in gradual denaturation of Hb and incorporation of the released Hb-iron into ferritin. Following treatment with APH, the ratio of Hb-57Fe to ferritin-57Fe decreased from 2.6 to 0.2 in MEL cells and from 0.56 to 0.12 in K-562 cells. No change was observed in the total intracellular iron. Using fluorescence ELISA, an increased level of immunologically detectable ferritin was found in hemoglobinized K-562 cells treated with APH, as compared to the amount of ferritin found in untreated cells. Ferritin may thus function not only as an intermediate during Hb synthesis, but also as storage protein for iron released during Hb denaturation.

Radioactively-labelled palmitic acid was used to study the effects of albumin, low temperature and several inhibitors of metabolism on transport of fatty acids into cultured human leukemic myeloid cells. When serum or albumin were present in the medium, uptake of fatty acid by cells as well as its further incorporation into phospholipids and neutral lipids were considerably reduced. Uptake and metabolic utilization of this fatty acid was reduced at low temperature, in the presence or absence of albumin in the incubation medium. In absence of albumin, addition of iodoacetate, sodium cyanide or sodium azide had but little effect on the total uptake of fatty acids while metabolic utilization was reduced. When albumin was present, these inhibitors reduced both total uptake and incorporation into lipids. The data suggest that incorporation of the fatty acid into the outer layer of the cell membrane is controlled by the concentration of free, uncomplexed molecules of fatty acid adjacent to the cell surface. In the absence of albumin this is a fast reaction which reaches nearly maximal uptake in three minutes. In the presence of albumin, this process is much slower and follows a nearly linear course between 3 and 60 minutes. Translocation into the inner layer of the membrane and subsequent utilization for metabolic processes is a much slower process, which seems to depend on the quantity of the fatty acid in the outer layer.

The relationship between the effects of two types of inducers on the maturation of a line of human promyelocytic cells (HL-60) was studied. The dual potentiality of these promyelocytes was demonstrated by the ability of isolated colonies to differentiate into granulocytes, following induction by dimethylsulphoxide (DMSO) or express properties specific to macrophages following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). The differentiation process involved an irreversible step which occurred 48 h after exposure to DMSO, and a few h after exposure to TPA. This implies that the presence of the inducer in the culture medium is no more required for completion of the differentiation programme. Removal of the inducer prior to this step resulted in reversal of all the inducer-mediated effects. During the period of non-commitment the specific pathway of maturation was still undetermined; cells in which differentiation was initiated by exposure to DMSO were able to develop into macrophages after substitution of DMSO by TPA. Moreover, pre-exposure to DMSO and other inducers of granulocyte differentiation resulted in higher sensitivity to TPA, as indicated by the cell response to low TPA concentrations and more rapid expression of macrophage specific properties. These results suggest that the early stages in HL-60 differentiation are probably common to the granulocyte and the macrophage pathways.

Studies on the effects of thalassemic mutations on gene function in vivo have clinical as well as scientific implications. Usually these studies have been performed on nucleated red blood cell (RBC) precursors normally present in bone marrow. Many patients with beta-thalassemia are splenectomized and may have high levels of nucleated RBC, orthochromatic normoblasts, in their peripheral blood (1-5% of total RBC). The possibility of exploiting these cells instead of bone marrow as a source for nuclear and cytoplasmic RNA for expression studies was investigated. A simple procedure was developed for enrichment for normoblasts in blood samples withdrawn from patients prior to transfusion. Globin transcripts were analyzed in RNA purified from 12 patients. Unspliced precursor beta-mRNA molecules were observed in a patient with beta o-thalassemia, homozygous for a mutation at the 5' IVS2 splice site of the beta-globin gene. Detailed analyses showed that his mature beta-mRNA was larger than normal, and that a cryptic 5' splice site, approximately 50 nucleotides downstream from the normal one, was utilized. We conclude that peripheral blood can be used as a reliable source of RNA for the analysis of the effects of beta-thalassemia mutations on gene expression and the relationship to the clinical condition. Moreover, this procedure facilitates the comparison of in vivo gene expression with the results obtained from DNA transfection experiments with cloned beta-thalassemia genes.

Mouse (MEL) and human (K-562) erythroleukemia cell lines can be induced to undergo erythroid differentiation, including hemoglobin (Hb) synthesis, by extra cellular hemin. In order to study the effect of extracellular hemin on intracellular ferritin and Hb content, we have used Mossabauer spectroscopy to measure the amount of 57Fe incorporated into ferritin or Hb and a fluorescent enzyme-linked immunosorbent assay (ELISA) to measure the ferritin protein content. When K-562 cells were cultured in the presence of a 57Fe source either as transferrin or citrate, in the absence of a differentiation inducer, all the intracellular 57Fe was detected in ferritin. When the cells were cultured in the presence of 57Fe-hemin, 57Fe was found in both ferritin and Hb. 57Fe in ferritin increased rapidly, and after 2 days it reached a plateau at 5 X 10(-14) g/cell. 57Fe in Hb increased linearly with time and reached the same value after 12 days. Addition of other iron sources such as iron-saturated transferrin, iron citrate, or iron ammonium citrate caused a much lower increase in ferritin protein content as compared to hemin. When K-562 cells were induced by 57Fe-hemin in the presence of 56Fe-transferrin, 57Fe was found to be incorporated in equal amounts into both ferritin and Hb. However, when the cells were induced by 56Fe-hemin in the presence of 57Fe-transferrin, 57Fe was incorporated only into ferritin, but not into Hb, which contained 56Fe iron. These results indicate that in K-562 cells, when hemin is present in the culture medium it is preferentially incorporated into Hb, regardless of the availability of other extra- or intracellular iron sources such as transferrin or ferritin. In MEL cells induced to differentiate by dimethylsulfoxide (DMSO) a different pattern of iron incorporation was observed; 57Fe from both transferrin and hemin was found to incorporate in ferritin as well as in Hb.

Proteolytic activity was measured in murine erythroleukemic 745 cell line grown in culture, before and after the addition of agents which promote differentiation. The 36,000 X g soluble fraction of the cells degraded [14C]globin with maximal activity at pH 3.6, while the insoluble fraction failed to degrade [14C]globin within a pH range of 2.5-9.0. The acid protease activity in the soluble fraction of the undifferentiated murine erythroleukemic cells increased during the first 2 days in culture and remained constant during the following 4 days. We suggest that this activity resides in the lysosomes since it migrates together with the lysosomal marker alpha-mannosidase on colloidal silica gradients, shows maximum activity at acid pH and is sensitive towards inhibition by pepstatin. Induced differentiation of the cells by dimethyl sulfoxide, butyric acid or hexamethylene bisacetamide was concomitantly associated with a marked reduction in protease activity and the accumulation of hemoglobin within the cells. In contrast, in a non-inducible variant of 745 cell line DMSO failed to affect proteolysis. It is suggested that in murine erythroleukemic cells changes in acid protease activity are associated with the cellular triggered by chemical inducers.

Normal human monocytes and macrophages, as well as in vitro human leukaemic promyelocytic cell line (HL-60) transformed into macrophage-like cells by 12-0-tetradecanoylphorbol-13-acetate (TPA) generate potent procoagulant activity (PCA) similar to tissue thromboplastin. In the present study, only mild PCA was detected in primary cultures of cells from the peripheral blood of patients with acute lymphatic leukaemia (ALL), acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML). After exposure to TPA, AML and CML cells assumed characteristics specific to monocytes and macrophages. Differentiation was associated with the generation of PCA. PCA was not found in ALL cells exposed to TPA. The PCA of TPA-induced macrophages derived from AML and CML cells resembled tissue thromboplastin and normal monocyte and macrophage PCA in several aspects: (a) accelerated clotting via the extrinsic coagulation pathway, (b) inhibition by concanavalin A and protection against lectin inhibition by methyl-alpha-D-mannopyranoside, (c) localization in the cell membrane. The capacity for PCA generation is additional evidence for the similarity between TPA-induced macrophages derived from AML and CML cells and normal human monocytes and macrophages.

The effect of the plant diterpenes, phorbol derivatives and mezerein, on differentiation of various human myeloid leukemic cells to macrophages was determined. The results indicate that, within the group of phorbol esters tested, a correlation exists between the potency of the compounds as inducers of differentiation and their reported potency as tumor promoters. However, mezerein and 12-O-retinoylphorbol 13-acetate, which promote tumors only weakly or not at all, were found to be efficient inducers. The efficiency of all the active phorbol derivatives, including the weak inducers, also known to be weak promoters, could be potentiated by pretreatment of the cells with retinoids, compounds which have been reported to inhibit tumor promotion. Similar results were obtained in 3 different established cell lines, as well as in short-term cultures of cells obtained from patients with acute myeloid leukemia. The results suggest that the activities of the diterpenes as tumor promoters and inducers of differentiation are not necessarily linked. Moreover, certain conditions which are unfavorable for tumor promotion may not affect or even potentiate induction of differentiation.

Murine erythroleukemia cells are induced to differentiate by 0.5-5 ng of actinomycin D per ml. Murine erythroleukemia cells cultured with actinomycin D prolong cell doubling time but achieve the same density after 5 days as cells without inducer. Actinomycin D causes over 95% of the cells to become benzidine-reactive. [(3)H]Actinomycin D uptake into DNA can be detected within 2 hr and reaches a maximum (approximately 0.1 pmol/10(6) cells) by 10-12 hr. It is estimated that about one out of 10(5) dG.dC pairs is bound to actinomycin D. Commitment to differentiation, assayed by transfer of cells to culture without inducer, was detected as early as 5 hr. Unlike Me(2)SO, which causes a transient prolongation in G(1) at about 15-20 hr, cells cultured with actinomycin D show a more sustained increase in the proportion of the cells in G(1). Globin mRNA accumulation was detectable by 19 hr in culture. Alteration in DNA stability in alkaline sucrose gradients was detected by 19 hr. Actinomycin D induces synthesis of Hb(maj) and Hb(min) in approximately equal amounts. A decrease in rates of synthesis of RNA, DNA, and total protein occurs in cells cultured with actinomycin D, as well as in cells cultured with Me(2)SO. No evidence for an early action of actinomycin D at the plasma membrane was obtained by measurement of changes in cell volume or (86)RbCl uptake. Taken together, the present results indicate that actinomycin D is a potent inducer of differentiation of murine erythroleukemia cells and suggest that the target of its effect may be at the level of DNA.

Mycobacterium lepraemurium infection of mice produces a chronic lethal disease that is characterized by massive accumulation of macrophages throughout the mononuclear-phagocyte system. We studied the influence of M. lepraemurium infection on the composition and function of the hematopoietic system. Medullary erythropoiesis was virtually abolished, as reflected by a small number of erythroid elements and a decrease in the number and frequency of erythroid progenitors in the bone marrow, together with reduced uptake of 59Fe into bone marrow hemin. On the other hand, erythropoiesis was observed in the spleen, as demonstrated by a large number of erythroid cells, a sixfold increase of 59Fe uptake, and a pronounced increase in the number of erythroid progenitors. A considerable increase of monocyte progenitors was observed in the spleen, and a more modest increase was observed in the bone marrow. This increase may be accounted for, at least in part, by greatly increased levels of macrophage-colony-stimulating factor in the serum of infected mice. Thus, M. lepraemurium infection produces important changes in the hematopoietic system, during the course of which the spleen becomes the major hematopoietic organ.

Differentiation of murine erythroleukaemia cells by various inducers involves a step of irreversible commitment, after which the presence of the inducer is not required for completion of the process. Some cells become partially committed and give rise to differentiated as well as undifferentiated progeny. Commitment occurs asynchronously; under suboptimal inducing conditions, such as low concentration of inducer or short duration of exposure, both committed and uncommitted cells co-exist. In the present study the growth of these subpopulations was compared. Murine erythroleukaemia cells were exposed to the inducer hexamethylene-bisacetamide for 24 hr, then the inducer was removed by washing and the rate of proliferation of committed and uncommitted cells was measured. Commitment was scored by cloning the cells in inducer-free semi-solid medium and determining the cellular composition of the colonies with respect to haemoglobin content. The results indicated that following removal of the inducer the rate of proliferation was retarded similarly for both committed and uncommitted cells. Partially committed cells disappeared rapidly due to assymetrical cell division into fully committed and uncommitted cells. Both committed and uncommitted cells resumed logarithmic growth at 53 hr, but while uncommitted cells continued this pace until saturation was achieved, committed cells stopped multiplying earlier as a result of terminal differentiation.

Serum erythropoietic activity was determined in 32 patients with beta thalassemia major and intermedia. Quantitation was performed by an in vitro bioassay using rabbit erythroid precursor cells (CFU-E) either by colony assay or by 3H-thymidine uptake. 20 polytransfused beta-thalassemic major patients had erythropoietic activity (mean 89.3 +/- 36 milliunits/ml) which was not significantly different (p greater than 0.2) from normal individuals (51.3 +/- 32 milliunits/ml). 12 untransfused patients with beta thalassemia intermedia were found to have comparable serum erythropoietic activity (p greater than 0.01). These levels were much lower than those found in patients with aplastic anemia who had a comparable degree of anemia. We have shown that the low EPO activity in thalassemic patients was not due to experimental conditions (excess of ferritin, low transferrin) nor to specific inhibitors appearing in this disease. No correlation was found between the erythropoietic activity and sex or other clinical parameters of the patients such as severity of the anemia, splenectomy, iron chelation or transfusion therapy. 4 young thalassemic children (1-2 yr of age) studied had high erythropoietic activity ranging from 661 to 5793 milliunits/ml--significantly different from normal children of the same age. It is suggested, therefore, that a decrease in serum erythropoietin levels develops during the course of the disease.

Phorbol ester tumor promoters produce a rapid increase in adhesiveness of murine erythroleukemia (MEL) cells. Following treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and other tumor promoters, these cells adhere to the surface of the culture dish or become agglutinated to each other. Structurally related compounds which are devoid of tumor promoting activity failed to induce agglutination of MEL cells. Pentamidine isethionate (PI) and tosylamide-phenylethyl-chloromethyl ketone, two known inhibitors of trypsin-like enzymes, prevent the phorbol esters-induced adherence and agglutination. A short exposure to TPA results in an increase in protease activity at the alkaline pH range. This TPA-induced proteolytic activity is inhibited by PI. Induction of erythroid differentiation by hexamethylene-bisacetamide is associated with a decrease in TPA-induced cell adhesion and TPA-induced proteolytic activity. Taken together, these results suggest the participation of an alkaline proteolytic activity in the membranal changes evoked by phorbol esters.

Incubation of cells with 12-(1-pyrene) dodecanoic acid (P12), a fatty acid to which a pyrene nucleus has been covalently linked, followed by irradiation with long-wave ultra-violet light (LUV) at 366 nm, resulted in cytotoxicity. Syntheses of macromolecules was significantly decreased after 30 min, while an accumulation of trypan-blue positive, non-viable cells was observed several hours following irradiation. Cloning of the irradiated cells in semi-solid medium showed an exponential dose-response survival curve. Above a threshold dose colony number decreased, although the rate of clonal development and the final size were not affected. The sensitivity of detecting rare surviving cells could be increased by incubating the irradiated cells for several hours in liquid culture followed by concentrating intact cells by gradient sedimentation. Using this procedure, one surviving clonogenic cell could be detected in 10(7) irradiated cells/dish. More than 10 min of irradiation at 773 microV/cm was required to photosensitize the population below detection by this method. The possibility was considered that colonies derived from cells surviving sub-maximal LUV doses represent clones that are resistant to photosensitization, a phenomenon attributed to either inability to take up or metabolize P12, or resistance to the radiation-induced toxicity. Analysis of P12 uptake in the surviving clonal populations showed no significant difference as compared to the parental population. The results suggest that surviving cells reflect a phenotypic heterogeneity caused by variation in the physiological state such as the respective position in the cell cycle and are not genetically resistant mutants.

Disseminated intravascular coagulation (DIC) is a frequent occurrence in acute promyelocytic leukemia (APL), especially after onset of chemotherapy. We have used a human promyelocytic leukemic established cell line (HL-60) and various other human leukemic cells to investigate the effect of cytotoxic drugs on generation of procoagulant activity (PCA). The results indicate that, unlike normal human peripheral blood monocytes and certain other cell types where PCA induction requires active mRNA and protein synthesis, in HL-60 cells, compounds such as actinomycin D, puromycin, and cytosine arabinoside and a variety of other cytotoxic agents, induced generation of a potent PCA. Although different in its mechanism of induction, this HL-60 cell PCA was similar, and may be identical, to mononuclear cell tissue factor. The PCA induction was rapid and preceded the lytic effect of the drugs. It was first detected on the outer cell surface but, following prolonged exposure to the drugs, upon lysis of the cells, it was also found in the extracellular medium. This in vitro effect mimics the development of DIC in patients with APL. The system may, therefore, serve as a model for the study of the cellular and molecular events associated with PCA generation by malignant promyelocytes and DIC occurrence in patients with APL and other malignancies.

The use of chromosome banding techniques has provided a valuable diagnostic tool in various malignancies. The application of these methods, however, is often restricted by a low yield of mitotic cells and the patient's unwillingness to comply with repeated bone marrow aspiration. In an attempt to promote mitotic activity of leukemic cells from the bone marrow and peripheral blood, we employed a new method based on culturing the cells in the presence of a conditioned medium derived from a human bladder carcinoma cell line (5637). In addition to colony stimulating factor, this conditioned medium contains a factor that is capable of stimulating leukemic myeloblast proliferation. Bone marrow and peripheral blood mononuclear cells from 58 patients with a variety of myeloid leukemias were cultured for 24 to 120 hours in the presence or absence of conditioned medium. These bone marrow cells showed a pronounced increase in the mitotic index (5- to 50-fold) as compared to unstimulated cultures, and a greater than 100-fold increase as compared to fresh, uncultured bone marrow cells. Analyzable metaphases could be obtained even in marrow samples in which direct or 24-hour G-banding techniques had failed to reveal metaphases. The effect observed on peripheral blood cells was even more dramatic because prior to culture no mitotic cells were detected, whereas up to 2% mitotic cells were found in conditioned medium-stimulated peripheral leukemic cells. Karyotype analysis of 36 out of the 58 leukemic patients has shown that the chromosome changes discovered in conditioned medium stimulated cells were identical to those found in unstimulated cells. New chromosome aberrations, attributable to the stimulation of growth by conditioned medium, were not found. The quality of the metaphases analyzed following conditioned medium stimulation was considerably better than that of unstimulated samples. Frozen cells, when cultured with conditioned medium, were also suitable for cytogenetic analysis. Thus, the use of this conditioned medium permits adequate cytogenetic analysis even in cases where such analysis was previously impossible.

The Friend virus-infected murine erythroleukemia cell can be induced to differentiate along erythroid cells in culture with various compounds, including dimethyl sulfoxide. DNA from murine erythroleukemia cells cultured with dimethyl sulfoxide shows a decrease in sedimentation rate in alkaline sucrose gradients after alkali lysis of the cells. These changes can be detected as early as 27 hr after the beginning of culture. Similar results are observed with DNA of the cells cultured with other inducers, butyric acid and dimethylacetamide, but not with DNA from a variant cell line resistant to induction with dimethyl sulfoxide. Ultraviolet irradiation, which is known to cause similar changes in the sedimentation rate of DNA in alkaline sucrose gradients, induces differentiation of the murine erythroleukemia cells. These studies suggest that alterations in DNA may be related to events involved in the induction of differentiation of murine erythroleukemia cells by dimethyl sulfoxide.

Recently, a novel approach has been used in the treatment of leukemia: induction of the leukemic cells to undergo terminal differentiation. Based on its in vitro ability to induce differentiation in several myeloid leukemic cell lines, retinoic acid (RA) has been applied clinically in cases of myelodysplastic syndromes and acute myeloid and promyelocytic leukemia. In the present study we have determined in detail the ability of RA to induce expression of granulocytic functions in a human promyelocytic leukemia cell line (HL-60) and compared it with that of dimethylsulfoxide (DMSO). Several granulocytic characteristics (phagocytosis, surface adherence and generation of free radicals in response to phorbol-ester) were induced to the same degree by both agents. Other normal neutrophil functions, including lysozyme accumulation, spontaneous migration, chemotactic activity toward zymosan-activated serum (containing C5a), the peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and spontaneous motility in semi-solid medium were induced by DMSO, but they were absent or incompletely expressed in RA-induced cells. In contrast, only RA induced migration toward leukotriene B4 (LTB4). Simultaneous treatment with RA and DMSO proved synergistic with respect to morphological maturation and several functions (e.g. NBT reduction), but complementary stimulation of other activities (e.g. chemotaxis, lysozyme content) could not be demonstrated. Furthermore, characteristics induced by DMSO (i.e., expression of C5a and FMLP receptors and accumulation of lysozyme) were inhibited by the addition of RA.(ABSTRACT TRUNCATED AT 250 WORDS)

When added to mouse neuroblastoma cultures, the potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) inhibits spontaneous neurite formation as well as that induced in response to serum deprivation, prostaglandin E1, 5-bromo-2'-deoxyuridine, and papaverine. Other tumor-promoting macrocyclic plant diterpenes also inhibit neurite formation, whereas nonpromoting diterpenes do not. Inhibition by TPA was reversible and was unrelated to toxicity.

Spontaneous and induced differentiation of murine erythroleukemia cells (strain 745A DS19 ) is reversibly inhibited by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent promoter of mouse skin carcinogenesis, and by other tumor-promoting macrocyclic plant diterpenes, but it is not by nonpromoting diterpenes. Twelve clones randomly isolated from this strain vary in their response to TPA. All clones are induced to differentiate by several compounds, the most potent of which is hexamethylene bisacetamide. In six clones TPA (100 ng/ml) caused greater than 90% inhibition of differentiation, as measured by the appearance of benzidine-reactive cells. In two clones cell differentiation was not inhibited by TPA even at concentrations as high as 1 microgram/ml. In four clones, differentiation was only partially inhibited (16 to 47%) by TPA. Clones resistant to TPA inhibition of differentiation were also resistant to structurally related tumor-promoting agents. The isolation of variant cell lines, sensitive and resistant to TPA, provides a tool for elucidating the mechanism of tumor promoter-mediated inhibition of cell differentiation.

There are three types of myeloid leukemic cells, IR+D, IR+D- and IR-D-. IR+D+ cells were induced to differentiate to granulocytes in mass culture in liquid medium by conditioned medium (CM) from cultures of lungs from mice injected with endotoxin. About 90% of the leukemic cells were induced to differentiate, 50% to mature granulocytes and 40% to intermediate stages. An efficient induction of granulocyte differentiation was also obtained with CM from primary cultures of rat embryo or human spleen and there was a lower activity with CM from various other sources. IR+D- cells were induced to differentiate to about 20% cells with intermediate stages but not to mature granulocytes; IR-D- cells could not be induced to differentiate to intermediate or mature stages. IR+D+ cells were induced to form intermediate stages of granulocyte differentiation, to phagocytose and to attach to the surface of the Petri dish, three days after incubation with CM. Optimum induction of mature granulocytes required six more days incubation with CM. Mature granulocytes induced from leukemic cells showed cytochemical properties and a morphology in the electron microscope similar to that of normal mature granulocytes. These induced granulocytes did not form leukemiac in animals or colonies in agar. The granulocytes induced from the myeloid leukemic cells, therefore, behaved like normal mature granulocytes.

Exogenous serine proteases were found to induce differentiation in human myeloid leukemic cells from either in vitro established long-term cell lines or in primary cultures of cells derived directly from patients with acute myeloid leukemia. Exposure of the human promyelocytic cell line HL-60 to trypsin, chymotrypsin, or elastase induced the appearance, within 3-6 days, of neutrophilic granulocytes defined by their morphology, their ability to reduce nitroblue tetrazolium, and their efficient phagocytosis of latex particles. Upon further incubation monocyte-like cells appeared. While these cells developed into fully mature macrophages other types of cells disappeared and on day 12 the culture consisted of a pure macrophage population. The inducing effect could be observed when the enzyme was presented alone, whereas a synergistic effect was noted when the protease was added in the presence of subthreshold concentrations of chemicals known to induce differentiation in this cell line such as dimethylsulfoxide, retinoic acid, butyric acid, or hexamethylene bisacetamide. Optimal induction of differentiation by trypsin required a 48 hr continuous exposure to the enzyme. When the protease was removed earlier no appreciable differentiation was noticed. The protease-induced differentiation involved a direct interaction with the cells and was not due to a proteolytic cleavage of a serum component because it could be obtained in serum-free cultures. The enzymatic activity of the protease was needed for its effect on cell maturation: Addition of protease inhibitors such as soybean-trypsin inhibitor or trasylol completely blocked differentiation induced by the proteases but had no effect on differentiation induced by the other inducers. It is still to be determined whether a proteolytic process is a general molecular event in cell differentiation or induction by chemicals involves a mechanism different from that initiated by exogenous proteases.

Cells from a myeloid leukemic line in culture can be induced by the differentiation-inducing protein MGI to form colonies with normal differentiation to mature macrophages and granulocytes. This line consisted of clones that can be induced to undergo normal cell differentiation (D(+) clones) and clones (D(-) clones) that were not inducible. D(+) clones were able to undergo differentiation to both macrophages and granulocytes. Normal differentiation was induced even in clones that were no longer diploid. D(+) clones can segregate some D(-) progeny, and D(-) clones can segregate some D(+) progeny. This, therefore, provides a system for studies on the genetic and chemical control of cell differentiation in leukemic cells.

Clones (D(+)) of a cultured line of myeloid leukemic cells can be induced to undergo normal differentiation to mature macrophages and granulocytes. There are also clones derived from the same cell line (D(-)) that could not be induced to differentiate. The carbohydrate-binding protein concanavalin A was used as a probe to study the mobility of carbohydrate-containing sites on the surface membrane of these cells. Changes in the distribution of concanavalin A binding sites on the surface membrane can be induced by concanavalin A. With the appropriate site mobility, this induction of a new distribution resulted in a concentration of concanavalin A-membrane site complexes on one pole of the cell to form a cap. D(+) and D(-) clones showed 50 and 5% of cells with caps, respectively, although both types of cells bound a similar number of concanavalin A molecules. Treatment of cells with trypsin increased cap formation from 5 to 40% in D(-) cells, but did not change the percentage of cells with caps in D(+) cells. The results show a difference in the mobility of concanavalin A binding sites in these two types of cells and suggest a difference in the fluid state of these carbohydrate-containing structures on the surface membrane. It is suggested that a gain of the ability of myeloid leukemic cells to undergo normal differentiation is associated with an increase in the fluidity of structures on the surface membrane where the concanavalin A sites are located. Differences in fluidity of specific membrane sites may also explain differences in the response of cells to other differentiation-inducing stimuli.