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D L Laskin

Publications and source records attributed to D L Laskin.

At least 73 records · Page 4Linked to original sources
PubMed1994

Pulmonary and hepatic effects of inhaled ozone in rats.

Nitric oxide is a highly reactive molecule that has been implicated in host defense and in tissue injury. In the present studies we analyzed the effects of brief exposure of rats to inhaled ozone on production of this mediator by lung macrophages and type II epithelial cells. We found that ozone exposure (1-2 ppm, 3 hr) induced a marked increase in spontaneous nitric oxide production by alveolar (AM) and interstitial macrophages, as well as type II cells. These effects were apparently due to increased expression of inducible nitric oxide synthase protein and mRNA, which was evident in vitro in isolated cells and in situ in histologic sections. Macrophages and epithelial cells from ozone-treated rats were also sensitized to produce increased amounts of nitric oxide in response to inflammatory cytokines such as interferon-gamma, a response that was also mediated by inducible nitric oxide synthase. Unexpectedly, we also discovered that brief inhalation of ozone caused dramatic effects on the liver, including increased production of nitric oxide by hepatocytes and enhanced protein synthesis. These data suggest that this inhaled irritant induces an acute phase response. Additional studies indicate that AM from ozone-treated rats produced significantly more tumor necrosis factor-alpha and interleukin-1 than did cells from control animals. Elevated levels of tumor necrosis factor-alpha were also noted immunohistochemically in both lung and liver tissue. These results indicate that the extrapulmonary effects of ozone may be mediated by inflammatory cytokines released by activated lung macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1994

Alterations in expression of p56lck during myeloid differentiation of LSTRA cells.

The src-related tyrosine kinase p56lck is overexpressed in the mouse leukemia cell line LSTRA. Although p56lck is thought to be a specific T-cell marker, we found that LSTRA cells can be induced to differentiate towards macrophages or granulocytes by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate or the cyclic nucleotide analogue, dibutyryl cAMP, respectively. Treatment of LSTRA cells with 12-O-tetradecanoylphorbol-13-acetate resulted in marked alterations in morphology including increased size, adherence, and spreading on culture dishes. These cells also ceased proliferating, accumulated in G0-G1 and expressed nonspecific esterase activity. In contrast, although LSTRA cells treated with dibutyryl cAMP stopped growing and accumulated in G0-G1, these cells expressed functionally active chemotactic peptide receptors and became irregular and granular in appearance. Differentiation of LSTRA cells was also found to be associated with altered expression of p56lck. Thus, while 12-O-tetradecanoylphorbol-13-acetate treatment caused the cells to produce higher molecular weight forms of p56lck, dibutyryl cAMP treatment resulted in increased expression of total p56lck mRNA as well as the more mature type II p56lck mRNA transcript. There were no major alterations in p56lck kinase activity in vitro following differentiation. Phospholipase C gamma and p21rasGAP, two putative substrates for the tyrosine kinase activity of p56lck, were found to be constitutively phosphorylated on tyrosine in LSTRA cells. Tyrosine phosphorylation of these substrates was not altered following differentiation. These results indicate that LSTRA cells are relatively early precursors that have the capacity to develop along the myeloid differentiation pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Differentiation↗
PubMed1994

Regulation of hepatic endothelial cell and macrophage proliferation and nitric oxide production by GM-CSF, M-CSF, and IL-1 beta following acute endotoxemia.

Treatment of rats with bacterially derived lipopolysaccharide (LPS), a condition that mimics acute endotoxemia, results in a significant increase in the number of endothelial cells and macrophages in the liver. This is correlated with the release of proinflammatory and cytotoxic mediators that induce liver damage. In the present studies, we analyzed the effects of various inflammatory mediators released during the pathogenesis of hepatic injury on proliferation of liver nonparenchymal cells. To induce acute endotoxemia female Sprague-Dawley rats were injected intravenously with 5 mg/kg LPS. Endothelial cells and macrophages were isolated 48 h later by combined collagenase and pronase perfusion of the liver followed by centrifugal elutriation. Interleukin-1 alpha (IL-1 alpha), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) had no effect on proliferation of either endothelial cells or macrophages. In contrast, whereas interleukin-1 beta (IL-1 beta) inhibited the proliferation of endothelial cells from untreated rats, this cytokine stimulated the growth of cells from endotoxemic rats. The colony-stimulating factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), also markedly enhanced the proliferation of endothelial cells, as well as macrophages from endotoxemic rats. Macrophages from endotoxemic rats were more sensitive to the colony-stimulating factors than cells from untreated rats. In contrast, the inflammatory mediators LPS and interferon-gamma (IFN-gamma) inhibited endothelial cell and macrophage growth, an effect that was partially blocked in endothelial cells by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). This suggests that growth inhibition in these cells is mediated, in part, by nitric oxide. Interestingly, in both endothelial cells and macrophages from endotoxemic rats, GM-CSF, M-CSF, and IL-1 beta synergized with LPS and IFN-gamma to induce nitric oxide production. This was correlated with a further inhibition of proliferation that was partially reversed by L-NMMA in endothelial cells but not macrophages. Taken together these data demonstrate that endothelial cell and macrophage proliferation in the liver is controlled by a variety of mediators released during endotoxemia; however, the mechanisms regulating growth in the two cell types are distinct.

Acute Disease↗
PubMed1993

Enhanced production of nitric oxide by rat alveolar macrophages after inhalation of a pulmonary irritant is associated with increased expression of nitric oxide synthase.

Alveolar macrophages represent the first line of defense of the lung against inhaled environmental agents. These cells release a variety of inflammatory mediators including reactive oxygen and nitrogen intermediates that have been implicated in host defense and in tissue injury. In the present studies we characterized production of these mediators by lung phagocytes after exposure of rats to an inhaled pulmonary irritant. Freshly isolated alveolar macrophages from control rats were found to produce nitric oxide as well as hydrogen peroxide and superoxide anion in response to in vitro treatment with inflammatory mediators such as IFN-gamma or LPS and phorbol esters, respectively. Production of nitric oxide by lung phagocytes was enhanced in the presence of superoxide dismutase. Western blot analysis revealed that production of nitric oxide after treatment of the cells with IFN-gamma and LPS was a result of increased expression of inducible nitric oxide synthase. After brief exposure of rats to ozone (O3, 1 to 2 ppm, 3 h), a pulmonary irritant and inflammatory agent that is rapidly converted to molecular oxygen, lung phagocytes produced significantly increased amounts of nitric oxide when compared with control animals. These cells were also sensitized to produce more nitric oxide in response to in vitro treatment with IFN-gamma and LPS. This was due, at least in part, to increased expression of inducible nitric oxide synthase by the cells, which was evident in protein blots and in immunohistochemically stained sections of lung tissue. In further studies we found that O3 inhalation also caused enhanced production of hydrogen peroxide, but an apparent decrease in release of superoxide anion by lung phagocytes. Taken together, these data demonstrate that acute irritant exposure modifies production of reactive oxygen and nitrogen intermediates by lung phagocytes. These alterations may be important in the pathophysiologic response of the lungs to irritants.

Amino Acid Oxidoreductases↗
PubMed1993

Induction of functionally active platelet-activating factor receptors in rat alveolar macrophages.

Freshly isolated alveolar macrophages from control rats express low numbers of platelet-activating factor (PAF) receptors (4.6 x 10(3) binding sites/cell, Kd = 1.3 nM). These receptors appeared to be functionally inert in that PAF failed to induce intracellular calcium mobilization in the cells. Following brief exposure of rats to ozone (2 ppm, 3 h), a pulmonary irritant and inflammatory agent that is rapidly converted to molecular oxygen, expression of PAF receptors was markedly up-regulated on lung phagocytes (13.1 x 10(3) binding sites/cell), with no significant effect on receptor affinity (Kd = 2.0 nM). In these cells, PAF, but not lyso-PAF, an inactive analog, caused a rapid and transient rise in intracellular calcium demonstrating that the PAF receptors were functionally active. The calcium response to PAF, which was largely due to mobilization of calcium from intracellular stores, was found to be dose-dependent in the nanomolar concentration range and inhibitable by the PAF receptor antagonist triazolam. The results of these studies may be important in elucidating mechanisms of lung inflammation induced by this model irritant.

Animals↗
PubMed1993

Characterization of interleukin-1 and interleukin-6 production by hepatic endothelial cells and macrophages.

Interleukin-1 (IL-1) and interleukin-6 (IL-6) derived from Kupffer cells are major inducers of hepatic inflammation and the acute phase response. The present studies demonstrate that liver endothelial cells also produce significant quantities of IL-1 and IL-6, suggesting that these cells also participate in these processes. Endothelial cells and macrophages were isolated from female Sprague-Dawley rats by combined collagenase and pronase perfusion of the liver followed by centrifugal elutriation. In the absence of stimulation, endothelial cells were found to spontaneously produce IL-1 and IL-6 in a time-dependent manner, reaching maximal levels after 10 h in culture for IL-1 and 6-8 h for IL-6. The amount and kinetics of cytokine production by hepatic endothelial cells were similar to those observed with Kupffer cells. In further studies, the effects of lipopolysaccharide (LPS), a potent liver macrophage activator and inflammatory agent, on cytokine release were analyzed. Treatment of rats with LPS resulted in a decrease in IL-1 release by both cell types compared to cells from untreated animals. In contrast, LPS treatment had no major effect on IL-6 release. We also found that both macrophages and endothelial cells could be induced to produce additional IL-1 and IL-6 by treatment with LPS in vitro, but only if they were preincubated for at least 24 h prior to stimulation with LPS and analyzed for cytokine release. These data demonstrate that liver endothelial cells, like Kupffer cells, have the capacity to produce immunoregulatory and proinflammatory cytokines.

Animals↗
PubMed1993

Platelet-activating factor-induced calcium mobilization and oxidative metabolism in hepatic macrophages and endothelial cells.

Fluorescence image analysis of the calcium sensitive dye Indo-1 was used to characterize platelet-activating factor (PAF)-induced calcium mobilization in hepatic macrophages and endothelial cells. PAF, but not lyso-PAF, an inactive analog, induced a rapid and transient increase in intracellular levels of calcium that appeared to depend on the presence of extracellular calcium. In both macrophages and endothelial cells, these effects were dose dependent, reaching maximal levels with 10 nM PAF. However, the kinetics of the calcium response to PAF in macrophages and endothelial cells was distinct. The endothelial cells responded more rapidly to PAF than the macrophages (within 1 min vs. 2-3 min, respectively) and displayed longer recovery periods (4 min vs. > 10 min, respectively). In contrast, the magnitude of the response to PAF was greater in the macrophages. In both cell types, triazolam and alprazolam, two PAF antagonists, and the calcium channel blocker verapamil inhibited PAF-induced calcium mobilization. PAF also stimulated superoxide anion production alone and in combination with the macrophage activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in both cell types. Macrophages produced more of this reactive oxygen intermediate in response to PAF and TPA than endothelial cells. Interestingly, in the absence of extracellular calcium or when verapamil was added to the cultures, superoxide anion production by the macrophages, but not the endothelial cells, was significantly reduced. These results demonstrate that, although hepatic macrophages and endothelial cells mobilize calcium in response to PAF, the characteristics of the response in each cell type are different. These differences may underlie, at least in part, distinct functional responses of the cells to PAF.

Alprazolam↗
PubMed1993

A single exogenous stimulus activates resident rat macrophages for nitric oxide production and tumor cytotoxicity.

Based on experiments with mouse cells, it appears that macrophage activation for cytotoxicity requires two exogenous signals. One signal primes or sensitizes the cells, while the second activates them for killing. The present studies were designed to analyze the capacity of rat macrophages to be activated for nitric oxide production and for cytotoxicity by different inflammatory stimuli. We found that both resident alveolar macrophages (AMs) and resident peritoneal macrophages (PMs) from Sprague-Dawley rats produced nitric oxide in response to relatively low doses of a single exogenous activating stimulus [interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS)]. Resident PMs treated with either of these agents alone also exhibited nitric oxide-mediated cytotoxicity toward xenogeneic and allogeneic tumor targets. In contrast to results reported previously with both resident and elicited PMs from mice, tumor necrosis factor-alpha (TNF-alpha) exerted only a small enhancing effect on IFN-gamma-induced nitric oxide production by resident rat PMs. In addition, although some level of cooperativity between IFN-gamma and LPS was observed at low concentrations of LPS (< 10 ng/ml), IFN-gamma did not augment the effects of higher concentrations of LPS (> or = 10 ng/ml) on nitric oxide production by PMs. In contrast to PMs, AMs had a strong synergistic response to combinations of IFN-gamma and LPS or TNF-alpha but also only at relatively low concentrations of IFN-gamma and LPS. Furthermore, maximum nitric oxide production induced by IFN-gamma in these cells could be enhanced by TNF-alpha or low doses of LPS. However, as observed with PMs, combinations of IFN-gamma and higher doses of LPS did not significantly augment maximum nitric oxide production induced in AMs by LPS alone. Thus, our data suggest that resident rat PMs and AMs resemble elicited mouse PMs in their ability to respond to a single activating stimulus. However, PMs and in some respects AMs differ from the latter by their reduced responsiveness to combinations of activators. Taken together, our results suggest that two exogenous stimuli are not required for full activation of resident macrophages from Sprague-Dawley rats.

Animals↗
PubMed1993

Isolation and partial characterization of subpopulations of alveolar macrophages, granulocytes, and highly enriched interstitial macrophages from rat lung.

The contribution of interstitial macrophages (IM) to lung defense, homeostasis, and pathophysiology is for the most part unknown. Studies on this cell type are difficult because they are not readily accessible in large numbers or in high purity. In the present work, various nonenzymatic and enzymatic methods were compared with the aim of isolating and characterizing pure populations of lung IM. The results of our studies demonstrate that most procedures currently used to isolate IM yield subpopulations of alveolar macrophages (AM) or IM highly contaminated by AM and granulocytes. We found that lavage of the lung yielded only one half of the total AM present in the tissue. The remainder of the AM could only be obtained by extensive washing of cut and disaggregated lung tissue, which is considered by some investigators to be an effective procedure for IM isolation. According to our results, cells recovered by lavage and washing of cut and disaggregated lung tissue were morphologically and histochemically identical, were strongly positive for nonspecific esterase, highly phagocytic, and appeared to represent subpopulations of AM with apparently varying degrees of adherence to the alveolar walls. We also found that IM could be obtained in high purity by sequential digestion of the remaining lung tissue with 60 and 175 IU/ml of collagenase followed by selective adherence. Digestion of the tissue with 60 IU/ml of collagenase resulted in a highly enriched population of granulocytes and also reduced their contamination in the IM population. The resulting IM were distinct from AM by morphology and histochemistry. Like AM, these cells displayed Fc receptor-mediated phagocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes. Potential role for nitric oxide in the regulation of wound healing.

In the skin, wounding initiates a complex array of physiological processes mediated by growth factors and inflammatory mediators which stimulate tissue repair and protect against infection. We report that primary cultures of human keratinocytes and a mouse keratinocyte cell line respond to the inflammatory stimuli gamma-interferon and lipopolysaccharide or tumor necrosis factor-alpha by producing nitric oxide and hydrogen peroxide, two reactive mediators that are important in nonspecific host defense. Nitric oxide is produced by the l-arginine- and NADPH-dependent enzyme, nitric oxide synthase. In murine keratinocytes, optimal enzymatic activity was found to be dependent on Ca2+ and calmodulin as well as on glutathione. Inflammatory mediators were also found to inhibit the growth of keratinocytes, an effect that could be reversed by a nitric oxide synthase inhibitor. Epidermal growth factor (EGF), which promotes wound healing by stimulating cellular proliferation, was found to be a potent antagonist of reactive nitrogen and reactive oxygen intermediate production by keratinocytes. EGF also reversed the growth inhibitory actions of the inflammatory mediators. These data suggest that nitric oxide produced by keratinocytes is important in the control of cellular proliferation during wound healing. Our findings that EGF effectively regulates the production of free radicals by keratinocytes may represent an important pathway by which this growth factor not only stimulates epidermal cell proliferation but also facilitates the resolution of inflammation following wounding.

Amino Acid Oxidoreductases↗
PubMed1992

Production of nitric oxide by murine bone marrow cells. Inverse correlation with cellular proliferation.

The present studies were designed to assess the ability of primary cultures of bone marrow cells to produce nitric oxide. We found that two inflammatory stimuli, IFN-gamma and LPS, were potent inducers of nitric oxide production by bone marrow cells. In addition, the CSF granulocyte-macrophage (GM)-CSF and IL-3 as well as TNF-alpha, while inactive by themselves, were synergistic with LPS and IFN-gamma in inducing nitric oxide production. Maximal effects were observed with combinations of GM-CSF and LPS. Nitric oxide production by bone marrow cells was found to be dependent on the presence of L-arginine in the culture medium and inhibitable by NG-monomethyl-L-arginine and L-canavanine, two nitric oxide synthase inhibitors. Nitric oxide produced by the cells was also suppressed by TGF-beta 1 and the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Separation of bone marrow cells by density gradient centrifugation and flow cytometry revealed that the granulocyte-containing fraction was largely responsible for nitric oxide production. In additional experiments we found that treatment of bone marrow cells with GM-CSF significantly stimulated bone marrow cell growth. In contrast, the combination of GM-CSF and LPS or IFN-gamma markedly suppressed cellular proliferation. This suppression was completely reversed by treatment of the cells with NG-monomethyl-L-arginine. Taken together, these data demonstrate that various inflammatory stimuli and cytokines induce nitric oxide production by primary cultures of bone marrow cells and that this mediator may play a role in the regulation of bone marrow cell growth and development.

Animals↗
PubMed1992

Lipopolysaccharide treatment of rats alters antigen expression and oxidative metabolism in hepatic macrophages and endothelial cells.

Endothelial cells and macrophages are located within the hepatic sinusoids. These two cell types play an important role in the clearance of bacterially derived lipopolysaccharide from the portal circulation. Our laboratory has previously demonstrated that treatment of rats with lipopolysaccharide results in the accumulation of macrophages in the liver that display properties of activated mononuclear phagocytes. This study was designed to analyze the effects of lipopolysaccharide on hepatic endothelial cells. Female Sprague-Dawley rats were treated with 5 mg/kg of lipopolysaccharide. Macrophages and endothelial cells were isolated from the rats 48 hr later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that lipopolysaccharide treatment of rats resulted in an increase in the number of both macrophages and endothelial cells recovered from the liver. Using specific monoclonal antibodies and flow cytometry, both macrophages and endothelial cells were found to express cell surface markers for Ia antigen, leukocyte common antigen, CD4 and the macrophage antigen, ED2. Macrophages expressed greater levels of these markers than endothelial cells. Flow cytometric analysis also revealed considerable subpopulation heterogeneity in the endothelial cells in antigen expression, physical characteristics and functional activity. Treatment of rats with lipopolysaccharide decreased expression of cell surface markers on the macrophages but not on the endothelial cells. This may be due to the distinct origin of these cells. To determine whether endothelial cells, like macrophages, were activated by lipopolysaccharide, we examined their ability to produce reactive oxygen intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Increased production of tumor necrosis factor-alpha by bone marrow leukocytes following benzene treatment of mice.

Hematopoiesis is regulated by cytokines released from bone marrow stromal cells and mature leukocytes. Recent studies have identified these cells as targets for benzene-induced hematotoxicity. In the present studies we analyzed the effects of benzene treatment of mice on the production of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) by bone marrow leukocytes. Bone marrow cells isolated from control or benzene-treated mice (660 mg/kg, once/day, 3 days) were purified on lymphocyte separation medium. Cells were then cultured in the presence of varying concentrations of lipopolysaccharide (0.1-10 micrograms/ml) for 0.5-48 hr. IL-1, IL-6, and TNF-alpha activity in culture supernatants was then quantified. We found a significant (p less than or equal to 0.02) increase in TNF-alpha production by bone marrow leukocytes from benzene-treated mice when compared to cells from control animals. Furthermore, this increase was dependent on the macrophage-specific growth factor, colony stimulating factor-1. Benzene treatment was also found to induce a small but significant (p less than or equal to 0.02) increase in the production of IL-1 by bone marrow leukocytes. This increase was rapid and transient, occurring in supernatants collected 2 hr after inoculation of bone marrow cells into culture. In contrast, benzene treatment had no effect on the production of IL-6 by bone marrow leukocytes. These results demonstrate that benzene treatment of mice stimulates mature bone marrow leukocytes to produce elevated levels of growth regulatory cytokines.

Animals↗
PubMed1992

Alterations in the morphology and functional activity of bone marrow phagocytes following benzene treatment of mice.

Benzene is a well-established hematotoxin that affects developing leukocytes and erythrocytes as well as bone marrow stromal cells. In the present studies we analyzed the effects of benzene on the morphology and functional activity of bone marrow phagocytes. Male Balb/c mice were treated with benzene (660 mg/kg) once per day for 3 days. Bone marrow cells were then isolated and fractionated by density gradient centrifugation. Using highly sensitive techniques in flow cytometry/cell sorting, we found that we could separate three distinct populations of bone marrow cells that differed with respect to size and density. Monoclonal antibody binding and cell sorting revealed a large, dense population that consisted predominantly of granulocytes, a smaller, less dense population of lymphocytes, and a population of intermediate size and density consisting of mononuclear phagocytes and precursor cells. Differential staining of sorted mononuclear phagocytes revealed that benzene treatment of mice caused a marked increase in the number of mature, morphologically activated macrophages in the bone marrow. Benzene treatment of mice also resulted in enhanced chemotaxis and production of hydrogen peroxide by bone marrow granulocytes and mononuclear phagocytes. In contrast, treatment of mice with the combination of hydroquinone and phenol (50 mg/kg each, 1 x/day, 3 days), two metabolites of benzene, resulted in a significant (p < or = 0.02) depression of granulocyte chemotaxis and had no effect on hydrogen peroxide production by bone marrow phagocytes compared to cells from control animals. Taken together these results demonstrate that benzene causes increased differentiation and/or activation of phagocytes in the bone marrow.

Animals↗
PubMed1991

Distinct patterns of chemotactic peptide-induced calcium mobilization in differentiated myeloid leukemia cells and peripheral blood neutrophils.

Flow cytometry was used to compare intracellular calcium mobilization in mature neutrophilic granulocytes (PMN) with HL-60 promyelocytic leukemia cells induced to differentiate with dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP). Using the calcium-specific probe indo-1 acetoxymethyl ester, we found that the ability of differentiating HL-60 cells to mobilize calcium in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) developed concomitantly with expression of receptors on the cells for this peptide. Mobilization of calcium in HL-60 cells, as well as in PMN, in response to fMLP was dose dependent and was related to the presence of calcium in the culture medium. In calcium-free medium, tenfold higher concentrations of fMLP were required to induce calcium mobilization than in calcium-containing medium. In HL-60 cells, calcium mobilization occurred more rapidly than in PMN and was independent of extracellular calcium. Furthermore, the response of HL-60 cells to fMLP was more prolonged than the response of PMN and was also of greater magnitude. Calcium mobilization in both HL-60 cells and PMN was partially inhibited by the calcium channel blocker, verapamil, but completely blocked by trimethylbenzoic acid 8-(diethylamino) octyl ester, an intracellular calcium antagonist. These results indicate that although both cell types mobilize calcium in response to fMLP, the characteristics of the responses are distinct. These differences may underlie distinct functional responses of PMN and differentiated HL-60 cells to fMLP.

Biological Transport, Active↗
PubMed1991

Distinct patterns of sulfated proteoglycan biosynthesis in human monocytes, granulocytes and myeloid leukemic cells.

The production of sulfated proteoglycans was compared in mature peripheral blood granulocytes and monocytes and HL-60 promyelocytic leukemia cells. We found that HL-60 cells synthesized 5-10 times more proteoglycans than peripheral blood leukocytes. Differentiation of HL-60 cells toward mature monocytes by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or towards granulocytes by treatment with retinoic acid or dibutyryl cyclic adenosine-3',5'-monophosphate (dbcAMP) resulted in a small (20-30%) decrease in sulfated proteoglycan biosynthesis. Chondroitin sulfate was found to be the predominant proteoglycan produced by monocytes, PMN and undifferentiated HL-60 cells. Differentiated HL-60 cells produced chondroitin sulfate as well as sulfated proteoglycans sensitive to nitrous acid degradation. Similar results were observed in TPA, dbcAMP and retinoic acid differentiated HL-60 cells indicating that the changes in proteoglycan biosynthesis observed were independent of the developmental pathway. Using specific monoclonal antibodies and flow cytometry, we also found that HL-60 cells and monocytes produced chondroitin-4-sulfate, chondroitin-6-sulfate and chondroitin-O-sulfate while PMN only produced chondroitin-4-sulfate. In addition, although there were no significant differences in antibody binding to undifferentiated and differentiated HL-60 cells, the tumor cells bound 5-20 times more of the antibodies than the peripheral blood leukocytes. Our data demonstrate that sulfated proteoglycan production by HL-60 cells is distinct from PMN and monocytes. In addition, the fact that differentiated HL-60 cells continue to synthesize larger amounts of the proteoglycans than the peripheral blood leukocytes indicates these cells have not completely matured.

Cell Line, Transformed↗
PubMed1991

Differentiation of HL-60 myeloid leukaemia cells is associated with a transient block in the G2 phase of the cell cycle.

The human promyelocytic leukaemia cell line HL-60 can be induced to differentiate towards mature granulocytes by treatment with dibutyryl cyclic adenosine-3',5'-monophosphate (dbcAMP). Differentiation begins within 16-24 h of treatment and is associated with a time- and dose-dependent accumulation of cells in the G0/G1 phase of the cell cycle with a concomitant decrease in the number of cells in the S and G2 + M phases. Using acridine orange staining, we found that the RNA content of the cells also decreased following differentiation. Stathmokinetic analysis of HL-60 cell populations following dbcAMP treatment showed no effect on the total number of cells in the G0/G1 or S phases, or the rate of progression of cells through these cell cycle compartments. In contrast, dbcAMP was found to induce a transient arrest of the cells in the G2 phase. We also found that differentiation induced by dbcAMP did not require progression of the cells through the cell cycle. Cells arrested in either G1/S by hydroxyurea or G2 + M by colcemid eventually expressed markers of mature granulocytes. These results demonstrate that dbcAMP modulates cell cycle progression. However, these cell cycle changes alone are insufficient to induce granulocytic differentiation of HL-60 cells.

Acridine Orange↗