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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.