Role of protein kinase C in phosphorylation of vinculin in adriamycin-resistant HL-60 leukemia cells

In response to phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), HL-60 cells differentiate to macrophage-like cells and exhibit the ability to phosphorylate vinculin in vitro. Adriamycin-resistant HL-60 (HL-60/ADR) cells similarly demonstrate this characteristic without prior treatment with TPA. Since protein kinase C (PK-C) is a cellular TPA receptor, we have examined the role of this enzyme in the inherent ability of HL-60/ADR cells to phosphorylate vinculin. DEAE-cellulose chromatography of cell extracts revealed that HL-60/ADR cells contained 2-fold more PK-C than did the parental cell line. All PK-C activity was found in the cytosol of wild type HL-60 cells, whereas 85% of PK-C activity was cytosolic and 15% was membrane-bound in HL-60/ADR cells. After a 2-day treatment with 10 nM TPA, PK-C activity was reduced 80-90% in both cell lines regardless of its intracellular distribution. Immunoblotting of cell extracts from HL-60/ADR cells or HL-60 cells following treatment with TPA revealed increased levels of a 52-kDa species of similar mass to M-kinase. Coincident with these changes after TPA treatment was a reduction in Ca2+ and phospholipid-independent phosphorylation of vinculin in vitro in extracts from HL-60/ADR cells, whereas HL-60 cells exhibited an elevation of this phosphoprotein. The phosphorylation of vinculin in TPA-treated HL-60 cells or untreated HL-60/ADR cells was blocked by antibodies to protein kinase C. These results suggest that it is not the absolute level of protein kinase C but rather the proteolytic activation of PK-C to a Ca2+ and phospholipid-independent form which is associated with the utilization of vinculin as an endogenous substrate.     

Decreased expression of type II protein kinase C in HL-60 variant cells resistant to induction of cell differentiation by phorbol diester

To evaluate the molecular basis for susceptibility of the cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), we examined biochemical activities and expression of subspecies of protein kinase C from HL-60 cells that are susceptible to differentiation induced by TPA and HL-60R cells, HL-60 variant cells that are resistant to such induction. Analysis of the subcellular distribution of protein kinase C revealed that the activity of this kinase in the cytosol from HL-60R cells was 30% of that from parental HL-60 cells but that the enzyme activities in the membrane showed similar values in these cells. Treatment of HL-60 cells with 100 nM TPA for 30 min resulted in a 75% decrease in protein kinase C activity in the cytosol and a 300% increase in this activity in the membrane. A minor subcellular redistribution of the enzyme activity was found in HL-60R cells after TPA treatment. Based on analysis of protein kinase C isozymes by hydroxyapatite column chromatography, the relative activities of types I, II, and III in the cytosol of HL-60 cells were 11, 80, and 9%, whereas those in HL-60R cells were 27, 36, and 37%, respectively. Type II isozyme was a major protein kinase C in the cytosol of HL-60 cells, but type II was less in the HL-60R cells. Among the three isozymes, type II enzyme was most sensitive to TPA with histone H1 as the substrate, although all three isozymes were activated Ca2+-dependently in the presence of phosphatidylserine. We suggest that the acquired resistance of HL-60R cells toward induction of cell differentiation by TPA may be associated with a decrease in the expression of the type II isozyme of protein kinase C.     

Synergistic stimulatory effect of 12-O-tetradecanoylphorbol-13-acetate and capsaicin on macrophage differentiation in HL-60 and HL-525 human myeloid leukemia cells

Our previous studies demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) had pharmacological activity for the treatment of myeloid leukemia patients. In the present study, we investigated the effects of TPA alone or in combination with capsaicin (8-methyl-N-vanillyl-6-nonenamide) on growth and differentiation in myeloid leukemia HL-60 cells and in a TPA-resistant HL-60 variant cell line termed HL-525. Treatment of HL-60 cells with TPA (0.16-1.6 nM) for 48 h resulted in concentration-dependent growth inhibition and cell differentiation (via the macrophage pathway). Capsaicin (5-50 microM) inhibited the growth of HL-60 cells in a concentration-dependent manner. Treatment of HL-60 cells with capsaicin alone only resulted in a small increase in the number of differentiated cells but treatment of the cells with TPA in combination with capsaicin synergistically increased differentiation. Moreover, inhibitors of protein kinase C (PKC), 7-hydroxystaurosporin (UCN-01; 100 nM) and chelerythrine (0.5 microM), significantly decreased HL-60 cell differentiation induced by the combination of TPA and capsaicin. These results suggest that PKC may be involved in HL-60 cell differentiation induced by TPA in combination with capsaicin. Capsaicin alone caused a very small increase in differentiation in the TPA-resistant HL-525 cells. However, treatment of HL-525 cells with combinations of TPA (0.16 nM) and capsaicin (10-50 microM) caused a strong synergistic increase in differentiation. Results from the present study suggest that a combination of TPA and capsaicin may improve the therapeutic efficacy of TPA and overcome resistance to TPA in some myeloid leukemia patients.     

Synergistic effects of clinically achievable concentrations of 12-O-tetradecanoylphorbol-13-acetate in combination with all-trans retinoic acid, 1alpha,25-dihydroxyvitamin D3, and sodium butyrate on differentiation in HL-60 cells.

Our recent studies demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) has pharmacological activity for the treatment of acute myelocytic leukemia patients. In the present study, we investigated the potential synergistic effect of all-trans retinoic acid (RA), 1alpha,25-dihydroxyvitamin D3 (VD3), and sodium butyrate (NaB) on TPA-induced differentiation in HL-60 human promyelocytic leukemia cells. The cells were treated once with these agents for 48 h or treated every 24 h for 96 h. Treatment of HL-60 cells once with TPA, RA, VD3, or NaB for 48 h resulted in concentration-dependent growth inhibition and cell differentiation. At clinically achievable concentrations, TPA (0.16 nM) increased the number of adherent cells and RA (0.1-1 microM) increased the number of nitroblue tetrazolium (NBT)-positive cells. The combinations of TPA (0.16 nM) with RA (0.1-1 microM), VD3 (1 nM), or NaB (100 microM) for 48 h synergistically increased differentiation as measured by the formation of adherent cells (P < or = 0.01). Moreover, cells treated with various combinations of low concentrations of TPA, RA, VD3, and NaB every 24 h for 96 h resulted in a further decrease in cell growth and an increase in differentiation. At clinically achievable concentrations, the strongest stimulation of differentiation was achieved in cells treated with a "cocktail" that combined TPA, RA, VD3, and NaB. The synergistic effect of combinations of TPA with RA or NaB at clinically effective concentrations on HL-60 cell differentiation suggests that the combination of these agents may improve the therapeutic efficacy of TPA for the treatment of acute promyelocytic leukemia (APL) patients. A differentiation "cocktail" that combines TPA, RA, VD3, and NaB may provide an even more effective strategy for improving the therapeutic efficacy of TPA and RA.     

Mimicry of bryostatin 1 induced phosphorylation patterns in HL-60 cells by high-phorbol ester concentrations

The bryostatins are a group of macrocyclic lactones isolated from the marine bryozoan Bugula neritina. Bryostatin 1, like the phorbol esters, activates protein kinase C; however, it partially inhibits the phorbol ester induced differentiation of the human promyelocytic leukemic cell line HL-60. We compared the phosphorylation response in HL-60 cells treated with phorbol 12,13-dibutyrate or bryostatin 1. Bryostatin 1 enhanced the phosphorylation of the same proteins as did typical concentrations (10(-8)-10(-9) M) of phorbol 12,13-dibutyrate. In addition, bryostatin 1 caused the appearance of 2 phosphorylated protein spots with molecular weights of 70,000 and pIs of 6.3-6.4. These latter phosphorylations were evident after a 30-min exposure to bryostatin 1 at 6 nM. Phorbol 12,13-dibutyrate concentrations of at least 600 nM, approximately 100-fold that necessary to induce differentiation, also induced the appearance of these phosphoprotein spots. The Mr 70,000 phosphoproteins were located in the ionic detergent-soluble cellular fraction which would contain the cytoskeletal proteins. Their phosphorylation was almost totally on serine residues. We speculate that phorbol esters at very high concentrations may more closely resemble bryostatin 1.     

The effects of lithium on the growth and phorbol ester (TPA) induced differentiation of two HL-60 sublines

Two sublines of a human promyelocytic cell line, HL-60, were used to study the effect of lithium on TPA (12-o-tetradecanoylphorbol-13-acetate) induced macrophage-like differentiation. Although these sublines, HL-60 M and HL-60 JE, had different growth rates, both showed enhanced proliferation when treated with 5 mM lithium (128 +/- 2 and 141 +/- 1% in comparison to controls after 5 days of incubation, respectively). Treatment of the sublines with TPA for 72 h resulted in macrophage-like differentiation (assessed by cell adhesion) of about 90% at 10 nM TPA in HL-60 JE, whereas a maximum of 50% at 100 nM TPA was obtained in HL-60 M. Differentiation was also confirmed by non-specific esterase activity. However, incubation of both sublines with TPA and 5 mM lithium revealed that lithium has little or no effect on the macrophage-like differentiation of the HL-60 cell line.     

Alterations in histone phosphorylation in HL-60 cells specific to monocytic differentiation

In order to examine the role of histone phosphorylation in regulation of the pathway of HL-60 cell differentiation, cells were labelled with [32P]phosphoric acid and histones fractionated by two-dimensional polyacrylamide gel electrophoresis. The monocytic inducer 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to specifically stimulate phosphorylation of histone H2B in a concentration-dependent manner. At a concentration of 100 mM, H2B phosphorylation was stimulated 2.3-fold after 4 h. A second monocytic inducer 1,25-dihydroxy-cholecalciferol (100 nM) also induced phosphorylation specifically in histone H2B. In contrast, the granulocytic inducers DMSO (1.5%) or retinoic acid (1 microM) did not increase phosphorylation in any histone species.     

Changes in the phosphorylation status of a 19 kD cytosolic protein are linked to the growth arrest of HL-60 cells.

A major 19 kD cytosolic protein (p19) has been described in a number of cell systems with respect to its rapid phosphorylation when protein kinase C is activated and has been proposed as a key substrate of this enzyme. Phosphorylation of p19 occurs when the growth of cells is affected by 12-O-tetradecanoylphorbol-13-acetate (TPA) and it has been proposed that increased phosphorylation of p19 relates to the cessation of cell growth. This study delineates precisely the relationship between p19 phosphorylation changes in the growth and differentiation status of cells. Changes in the levels of two phosphorylated forms of p19 were assessed in HL-60 promyelocytic cells and a variant HL-60 cell line which stopped growing and differentiated in response to TPA and were compared to changes seen in HL-60 variant lines which merely growth arrested when treated with TPA. In lines which either did or did not differentiate, in response to TPA, the p19 protein was rapidly and transiently phosphorylated. Thus, this alteration in the phosphorylation status of p19 is associated with the process of growth arrest and not related to the onset of cell differentiation. The p19 protein and the enzymes which effect its phosphorylation status modulate the growth of cells and possible disregulation of p19 and/or its kinases and phosphatases is of interest as regards the leukaemic transformation of cells.     

Molecular mechanism involved in TPA-induced myelopoietic programming

The study, addressed to understand the mechanism responsible for 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced myeloid differentiation, revealed that the inherent capacity of TPA to induce expression of Receptor-C(K) in HL-60 cells (which are unable to express Receptor-C(K) in their native state) is responsible for its ability to induce phenotypic differentiation of HL-60 cells. Based upon these results, we propose that Receptor-C(K) dependent signalling is the Critical 'Molecular Switch' responsible for TPA-induced myelopoietic programming.     

Ribosomal p70S6K basal activity increases upon induction of differentiation of myelomonocytic leukemic cell lines HL60, AML14 and MPD

The role of ribosomal p70S6K in the cell cycle has been studied extensively, and it is known that this enzyme is crucial for cell advancement through G(1). Conversely, the participation of p70S6K in cell differentiation is not well understood. We have studied the response of p70S6K to the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) in three differentiation-capable leukemic cell lines (MPD, AML-14 and HL-60) and in normal mature neutrophils. Immature leukemic cells starved for 16 h showed a robust ( approximately 3.5-fold over controls) p70S6K phosphorylation on T(421)/S(424) residues in response to an acute (5 min) 10 nM GM-CSF stimulation. On the other hand, cells that had been induced to differentiate and express granulocytic phenotypes, showed an increased ( approximately 6-fold) basal level of p70S6K T(421)/S(424) phosphorylation over immature cells, as well as an increased baseline tyrosyl phosphorylation of the GM-CSF receptor beta subunit (GM-CSF.Rbeta). However, the differentiated cells displayed a weak ( approximately 1.4-fold over controls) response to GM-CSF even at prolonged incubation times (20 min). In vitro p70S6K enzymatic activity paralleled p70S6K T(421)/S(424) phosphorylation in both high basal, unstimulated, levels in immature cells and a low degree of response to GM-CSF. Lastly, peripheral blood mature neutrophils had low basal GM-CSF.Rbeta and p70S6K activity, with both parameters being robustly stimulated following addition of GM-CSF, a situation in contrast with the cell lines, indicative perhaps of their incomplete terminal differentiation. In summary, these findings show the increase in basal phosphorylation of p70S6K upon granulocytic differentiation of myeloid leukemic cells and their responses to GM-CSF that are closely paralleled with tyrosyl phosphorylation of its receptor, and help in pointing to specific cell signaling molecules that are different in leukemic blasts from normal leukocytes.     

Quaternary ammonium analogs of ether lipids inhibit the activation of protein kinase C and the growth of human leukemia cell lines

Purpose: ET-18-OCH₃ (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) is a representative of the first generation of antitumor ether lipids and is a model in the development of new compounds including a series of quaternary ammonium analogs (QAA). In the present study, we evaluated the QAA as inhibitors of cell growth and studied their mechanism of action. Methods: We compared the effects of the QAA on the proliferation of human leukemia cell lines which are sensitive (HL-60) or resistant to ET-18-OCH₃ (HL-60R and K562). In addition we used cell fractionation and enzymatic assays to determine the effects of QAA on protein kinase C (PKC) translocation in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Results: The QAA and ET-18-OCH₃ were approximately equally effective inhibitors of HL-60 cell growth. However, the QAA were more effective inhibitors of K562 and HL-60R cell proliferation. The HL-60R cells, which were selected for resistance to ET-18-OCH₃, were also resistant to BM 41.440 which is structurally similar. In serum-free medium, the phosphorus-containing compounds (ET-18-OCH₃, BM 41.440 and He-PC) were much more effective inhibitors (8–20-fold) of the K562 cell line while the activities of the QAA were only moderately increased (1.2–2.3-fold). When serum albumin was added to the serum-free medium, the K562 cells became resistant to ET-18-OCH₃, suggesting that albumin is responsible for the differential sensitivity. The QAA compounds, which inhibit PKC activity in vitro, inhibited cell proliferation. However, a QAA which did not inhibit PKC did not inhibit cell proliferation. The phorbol ester TPA stimulates PKC translocation and causes HL-60 cell differentiation to adherent macrophage-type cells. The QAA inhibited TPA-induced cell differentiation and PKC translocation indicating that they also inhibit PKC in intact cells. Conclusions: The cellular effects of the QAA appear to be due to inhibition of PKC. In addition, these data indicate that albumin, which is important as a mediator of the uptake of ET-18-OCH₃, has only a small effect on the uptake of QAA. Together these data indicate that the QAA are potential anticancer agents, showing a significant ability to inhibit growth of leukemia cell lines that are resistant to ET-18-OCH₃. Received: 1 July 1997 / Accepted: 25 November 1997     

Detection and characterization of membrane protein changes in multidrug resistant HL-60 cells.

Antisera were prepared against fractionated membrane proteins of HL-60 cells isolated for resistance to adriamycin. Analysis of these antisera revealed that one (GSBl) was capable of detecting major protein changes in three independent isolates selected for anthracycline resistance. Thus, in studies using western blot analysis, the antiserum was found to be reactive with two proteins of 130 and 150 kDa which are present in plasma membranes of resistant but not sensitive cells. The antibody also reacted with a plasma membrane protein of 180 kDa that is present in sensitive cells but is increased in resistant isolates. Additional studies showed that P180 was greatly increased in both plasma membranes and endoplasmic reticulum in sensitive cells induced to differentiate in the presence of 12-O-tetradecanoylphorbol13-acetate (TPA). Resistant cells treated under identical conditions showed only a slight increase in the levels of P180. TPA had no effect on the levels of P150 or P130. In contrast, differentiation of HL-60 cells in the presence of dimethylsulfoxide (DMSO) resulted in the induction of P150 expression with major levels of protein contained in plasma membranes. DMSO has essentially no effect on the levels of plasma membrane P180, P150, or P130 in HL-60/Adr cells. These results therefore demonstrate a strong correlation between the development of resistance and the overexpression of proteins reactive with the GSBl antiserum. The results also show that development of anthracycline resistance in HL-60 cells results in the overexpression of P150, a protein associated with the differentiation of myeloid cells to granulocytes.     

Effects of okadaic acid and vanadate on TPA-induced monocytic differentiation in human promyelocytic leukemia cell line HL-60

Treatment of HL-60 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) (1–5 nM) induced inhibition of cell growth and the appearance of an adherent monocyte-like cell type in a dose- and time-dependent manner. The extent of TPA-induced monocytic differentiation was found to be markedly reduced by okadaic acid (OA) (35 nM). OA had to be present for the early 12 h during treatment with TPA to reduce the induction of monocytic differentiation. The majority of cells (80%) were non-adherent but morphologically resembled mature myelocytes or granulocytes after treatment with TPA (5 nM) in the presence of OA (35 nM). Vanadate (VD), on the other hand, enhanced the extent of monocytic differentiation induced by low-dose of TPA (1 nM). These results indicated that dephosphorylation by tyrosine protein phosphatase and serine-threonine protein phosphatase may play an important role in the induction of monocytic and granulocytic differentiation.     

Retinoic acid promotes phorbol ester-initiated macrophage differentiation in HL-60 leukemia cells without disappearance of protein kinase C

The ability of retinoic acid (RA) to promote 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-initiated macrophage differentiation was examined in human promyelocytic leukemia cell line HL-60. One-hour exposure to 10 nM TPA and subsequent exposure for 48 h to 1 μM RA following removal of TPA rapidly induced the macrophage phenotype in 65% of the cells. This effect was comparable to continuous exposure for 48 h to TPA alone, but contrasted with the absence of macrophage-like cells after RA treatment alone or the induction of 10% of the cell population to a macrophage phenotype after 1-h exposure to TPA. The effect of TPA + RA was accompanied by increased cell adherence and increased nonspecific esterase activity but not by a change in the reduction of nitroblue tetrazolium. Protein kinase C (PK-C) activity was increased 35–40% in cells treated for 1 h with TPA alone or after subsequent exposure to RA. Cells treated for 48 h with RA exhibited a 2-fold increase in PK-C activity while cells exposed to TPA for 48 h lost all PK-C activity. The changes in PK-C activity in TPA + RA-treated cells were accompanied by Ca²⁺/phospholipid(PL)-dependent phosphorylation in vitro of pp38 which is characteristic of treatment with RA alone, as well as the Ca ²⁺/PL-independent phosphorylation in vitro of pp82 and pp130 (vinculin) which is prevalent in cells treated continuously with TPA alone and is absent in RA-treated cells. These results indicate that the macrophage phenotype induced by TPA + RA is similar to that produced by continuous exposure to TPA alone with respect to their in vitro phosphoprotein patterns, cytochemical markers, cell adherence and morphology, but that the disappearance of PK-C is not an obligatory characteristic of these cells.     

Changes in actin and actin-binding proteins during the differentiation of HL-60 leukemia cells.

Actin and actin-binding proteins form a peripheral network on the cytosolic side of the plasma membrane. These cytoskeleton proteins are involved in functions that require cellular movement and may also have a role in modulating signal transduction during cellular proliferation and differentiation. To measure changes in F-actin and actin-binding proteins during HL-60 differentiation, cells were induced to mature along the granulocytic pathway by exposure to 1 microM retinoic acid (RA) for 5 days and were analyzed for F-actin and actin-binding proteins by flow cytometry. The amounts of F-actin and spectrin in untreated HL-60 cells and in those undergoing differentiation by treatment with the retinoid did not differ. N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin was used to measure F-actin content and a monoclonal antibody followed by fluorescence isothiocyanate-conjugated goat anti-mouse immunoglobulin antibody was used to measure the content of spectrin; cells were analyzed by flow cytometry. In contrast, cells exposed to RA contained larger amounts of alpha-actinin, vinculin, talin, lipocortin I, and lipocortin II, as determined with their respective antibodies followed by flow cytometric analysis as described above. An RA-supersensitive clone of HL-60, designated HL-60/S4, exhibited lower constitutive levels of alpha-actinin, vinculin, and talin but a higher constitutive level of lipocortin II than parental cells. Treatment of HL-60/S4 with RA led to increases in vinculin, talin, lipocortin I, and lipocortin II. An RA-resistant clone, designated HL-60/R3, constitutively expressed larger amounts of alpha-actinin, vinculin, lipocortin I, and lipocortin II than parental HL-60 cells. Treatment of HL-60/R3 with RA resulted in decreases in the amounts of these actin-binding proteins. Changes in actin-binding proteins that occur during the differentiation of HL-60 cells suggest that these proteins may be of importance to the expression of the mature phenotype.     

HL-60 cells isolated for resistance to vincristine are defective in 12-O-tetradecanoylphorbol-13-acetate induced differentiation and the formation of a functional AP-1 complex.

HL-60 cells isolated for resistance to vincristine are multidrug resistant and defective in the cellular accumulation of drug. Further studies demonstrate that these cells are also highly defective in 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation to macrophages. Analysis of this system demonstrates that certain protooncogenes which may contribute to differentiation are expressed at similar levels in sensitive and resistant cells. Thus, treatment of cells with TPA results in a reduction in the levels of c-myb and c-myc mRNA, while the expression of c-fos, c-jun, and junB is greatly enhanced. Immunoprecipitation experiments also demonstrate a TPA induced increase in the c-jun protein in both sensitive and resistant cells. Gel mobility shift assays show that TPA induces AP-1 formation in sensitive cells, whereas in parallel experiments with the HL-60/Vinc isolate, AP-1 is essentially absent. It has been found, however, that in resistant cells which have reverted to drug sensitivity, the levels of TPA inducible AP-1 is essentially identical to that of sensitive cells. Revertant and sensitive cells differentiate at similar levels in the presence of TPA. These studies therefore demonstrate that HL-60/Vinc cells are defective in the TPA induction of a functional AP-1 complex and that this may account for the inability of these cells to differentiate to macrophages. The molecular basis of the finding that AP-1 is not formed in resistant cells remains to be determined.