Promyelocytic Leukemia Cell Line HL-60 Research Articles

OVCA2 is downregulated and degraded during retinoid-induced apoptosis.

Retinoids, the natural and synthetic derivatives of vitamin A, have been shown to regulate the growth and differentiation of a wide variety of cell types and consequently have enormous potential as chemotherapeutic agents. We have previously identified 2 genes, termed OVCA1 and OVCA2, which are located in a small region showing a high frequency of allelic loss in breast and ovarian tumors and share a common exon. Recent studies have suggested that expression of OVCA1 may be influenced by retinoids. Therefore, we analyzed the expression of OVCA1 and OVCA2 in cells in response to treatment with all-trans retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4HPR), or under conditions of low serum and confluence, to determine further the roles of OVCA1 and OVCA2 in cell growth, apoptosis and differentiation. We show that OVCA2 mRNA and protein are ubiquitously expressed and that they are downregulated in the lung cancer cell line Calu-6 after treatment with RA and 4HPR. In addition, we observed that OVCA2 protein is proteolytically degraded in response to RA and 4HPR treatment in a time- and dose-dependent manner in the promyelocytic leukemia cell line HL60. In contrast, expression of the candidate tumor suppressor OVCA1 was not downregulated by these treatments. Furthermore, we demonstrate that OVCA2 is evolutionarily conserved and shows regional homology with dihydrofolate reductases (DHFRs), specifically with hydrolase folds found in alpha-beta hydrolases. Our results are in contrast to a previous report and show that OVCA2, not OVCA1 mRNA and protein, is downregulated in response to RA and 4HPR.

Amifostine differentially modulates DNA damage evoked by idarubicin in normal and leukemic cells

Human lymphocytes, p53 protein-deficient acute promyelocytic leukemia cell line HL-60, murine pro-B lymphoid cell line BaF3 and its TEL/ABL-transformed clone cells were exposed to idarubicin with and without pre-treatment with amifostine. Idarubicin at 0.5–5 μM evoked DNA damage measured by the Comet assay. Amifostine at 14 mM decreased DNA-damaging effect of idarubicin in human lymphocytes and BaF3 cells, but increased the effect in TEL/ABL-transformed cells. Amifostine had no influence on the action of idarubicin in HL-60 cells. Our results suggest that the reaction of the cell to DNA damage may contribute to its diverse response to amifostine combined with anticancer drugs and that p53 and fusion tyrosine kinases may be involved in this diversity.

Proliferating or Differentiating Stimuli Act on Different Lipid-dependent Signaling Pathways in Nuclei of Human Leukemia Cells

Previous results have shown that the human promyelocytic leukemia HL-60 cell line responds to either proliferating or differentiating stimuli. When these cells are induced to proliferate, protein kinase C (PKC)-beta II migrates toward the nucleus, whereas when they are exposed to differentiating agents, there is a nuclear translocation of the alpha isoform of PKC. As a step toward the elucidation of the early intranuclear events that regulate the proliferation or the differentiation process, we show that in the HL-60 cells, a proliferating stimulus (i.e., insulin-like growth factor-I [IGF-I]) increased nuclear diacylglycerol (DAG) production derived from phosphatidylinositol (4,5) bisphosphate, as indicated by the inhibition exerted by 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and U-73122 (1-[6((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), which are pharmacological inhibitors of phosphoinositide-specific phospholipase C. In contrast, when HL-60 cells were induced to differentiate along the granulocytic lineage by dimethyl sulfoxide, we observed a rise in the nuclear DAG mass, which was sensitive to either neomycin or propranolol, two compounds with inhibitory effect on phospholipase D (PLD)-mediated DAG generation. In nuclei of dimethyl sulfoxide-treated HL-60 cells, we observed a rise in the amount of a 90-kDa PLD, distinct from PLD1 or PLD2. When a phosphatidylinositol (4,5) bisphosphate-derived DAG pool was generated in the nucleus, a selective translocation of PKC-beta II occurred. On the other hand, nuclear DAG derived through PLD, recruited PKC-alpha to the nucleus. Both of these PKC isoforms were phosphorylated on serine residues. These results provide support for the proposal that in the HL-60 cell nucleus there are two independently regulated sources of DAG, both of which are capable of acting as the driving force that attracts to this organelle distinct, DAG-dependent PKC isozymes. Our results assume a particular significance in light of the proposed use of pharmacological inhibitors of PKC-dependent biochemical pathways for the therapy of cancer disease.

Induction of Apoptosis with an Extract of Actinidia polygama Fruit in the Promyelocytic Leukemia Cell Line HL-60.

Common small fruit produced naturally in Japan is attracted as healthy food. We studied on inhibitory activity of several small fruit extracts on growth of cancer cells to look for a candidate of healthy food or low toxic anti-cancer drugs. Among the fruit extracts examined, Actinidia polygama (Matatabi) extract had dose-dependent potent inhibitory activity against the growth of a promyelocytic leukemia cell line, HL-60, but was less effective on a colon cancer cell line, LS-174T, or normal human dermal fibroblast cells. We examined a part of the responsible mechanism of growth inhibition of HL-60 cells. Active agents in A. polygama fruit were heat-stable and small molecular weight of less than 1000. HL-60 cells incubated with an extract of A. polygama fruit induced apoptosis, which was detected by electrophoretic DNA fragmentation, morphological chromatin condensation and the TUNEL method with flow cytometry.

PHOSPHATIDYLINOSITOL 3-KINASE REGULATES PMA-INDUCED DIFFERENTIATION AND SUPEROXIDE PRODUCTION IN HL-60 CELLS

ABSTRACTTreatment of the human promyelocytic leukemia cell line HL-60 with phorbol myristate acetate (PMA) is associated with induction of monocytic or myelocytic differentiation. Since phosphatidylinositol 3-kinase (PI3-kinase) is a critical player in cell proliferation, survival, and differentiation, we studied the role of PI3-kinase during induction of the differentiated monocytic phenotype and superoxide production. In treatment of HL-60 cells with PMA, the PI3-kinase inhibitors LY294002 and wortmannin inhibited cell adhesion and spreading and phagocytic activity. LY294002 and wortmannin also inhibited the proliferation of HL-60 cells. During PMA-induced monocytic differentiation, LY294002 induced apoptosis in a dose dependent manner. The phosphorylation of p85α derived from PMA-stimulated HL-60 cells was shown in the time dependent manner. However, p70 S6 kinase inhibitor, rapamycin, did not inhibit PMA-induced monocytic differentiation. During PMA-induced monocytic differentiation, LY294002 inhibited c-jun protein expression and decrease of c-myc protein level. In contrast, LY294002 induced production of superoxide in the HL-60 cells stimulated with forskolin. Moreover, staurosporine and H7, PKC inhibitors, enhanced superoxide production in dibutyryl cAMP-induced HL-60 cells. These results suggest that PI3-kinase may regulate PMA-induced differentiation signal and provide a crucial link between PKC and cAMP in HL-60 cells.

NoBP, a nuclear fibroblast growth factor 3 binding protein, is cell cycle regulated and promotes cell growth.

Secreted and nuclear forms of fibroblast growth factor 3 (FGF3) have opposing effects on cells. The secreted form stimulates cell growth and transformation, while the nuclear form inhibits DNA synthesis and cell proliferation. By using the yeast two-hybrid system we have identified a nucleolar FGF3 binding protein (NoBP) which coimmunoprecipitated and colocalized with FGF3 in transfected COS-1 cells. Characterization of the NoBP binding domain of FGF3 exactly matched the sequence requirements of FGF3 for its translocation into the nucleoli, suggesting that NoBP might be the nucleolar binding partner of FGF3 essential for its nucleolus localization. Carboxyl-terminal domains of NoBP contain linear nuclear and nucleolar targeting motifs which are capable of directing a heterologous protein beta-galactosidase to the nucleus and the nucleoli. While NoBP expression was detected in all analyzed proliferating established cell lines, NoBP transcription was rapidly downregulated in the promyelocytic leukemia cell line HL60 when induced to differentiate. Analysis on the expression pattern of NoBP mRNA throughout the cell cycle in HeLa cells synchronized by lovastatin demonstrated a substantial upregulation during the late G(1)/early S phase. NoBP overexpression conferred a proliferating effect onto NIH 3T3 cells and can counteract the inhibitory effect of nuclear FGF3, suggesting a role of NoBP in controlling proliferation in cells. We propose that NoBP is the functional target of nuclear FGF3 action.

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells.

The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

Synthesis and biological activity of glycosyl conjugates of N-(4-hydroxyphenyl)retinamide

Three glycosyl (glucosyl, galactosyl, mannosyl) conjugates of 4-hydroxyphenylretinamide have been synthesized and tested on a broad variety of tumor cells. All three compounds are active on promyelocytic leukemia cell lines HL60 but less than the parent compound 4-HPR. Among them, the mannosyl analog stands out by its very low toxicity.

Interactions Between P-Glycoprotein and Drugs Used in the Supportive Care of Acute Myeloid Leukemia Patients

Multidrug resistance due to overexpression of P-glycoprotein (Pgp) leads to reduced intracellular drug accumulation and makes the cells resistant to chemotherapy. In this study we focused on how drugs used in the supportive care of acute myeloid leukemia (AML) patients interfere with Pgp. The effect on intracellular accumulation of the fluorescent dye Rhodamine 123 (Rh 123) was studied in the human promyelocytic leukemia cell line HL-60 and two anthracycline resistant, Pgp expressing, sublines. Each drug was used at two different concentrations: plasma peak concentration and half the plasma peak concentration. Drugs which increased the Rh 123 uptake by >10% were included in the second part of the study where the cytotoxic effect was tested in combination with daunorubicin. In the Rhodamine assay none of the tested drugs had any significant effect on the Rh 123 efflux in the resistant cell lines. Amphotericin B, cefuroxime, erythromycin and dixyrazin had minor effects on Rh 123 uptake but showed a significant additive effect to the toxicity of daunorubicin suggesting other mechanisms of action than reversal of Pgp. In conclusion this in vitro model where Rh 123 uptake was studied in an anthracycline resistant leukemia cell line could not demonstrate any significant interactions with Pgp for the tested drugs.

The biological function of a fragment of the neurotrophic factor from pigment epithelium: Structural and functional homology with the differentiation factor of the HL-60 cell line

It was shown that the full-size neurotrophic factor from pigment epithelium (PEDF) induces the cell differentiation of the human promyelocyte leukemia cell line HL-60. A structural analysis of PEDF revealed in itsC-terminal region a six-membered peptide fragment PEDF-(352-357) (PEDF-6) whose sequence is highly homologous to the 41–46 fragment of the active site of the human leukocyte differentiation factor HLDF (HLDF-6). The biological effect of PEDF and synthetic peptides PEDF-6 and HLDF-6 on the HL-60 cells and the early gastrula ectoderm ofXenopus laevis embryos was studied. On the basis of the structural and functional homologies of HLDF, PEDF, and their homologous peptides and the computer models of the spatial structures of the full-size PEDF and the PEDF with theC-terminal fragment split off tby the cleavage of the Leu380-Thr381 bond in the serpin loop, a hypothesis on the functional role of the serpin loop in PEDF was put forward.

Susceptibility to apoptosis is restored in human leukemia HCW-2 cells following induction and stabilization of the apoptotic effector Bak

We demonstrate that treatment of HCW-2 cells, an apoptotic resistant variant of the human HL-60 promyelocytic leukemia cell line with phorbol-12-myristate acetate (PMA), induced differentiation along the monocytic lineage. During this process there was a dramatic increase in the mitochondrial levels of the apoptosis effector, Bak, due to the stabilization of bak mRNA, which was correlated with the sensitization of HCW-2 cells to respond to the apoptotic effect of staurosporine (STS). Treatment of PMA-differentiated, but not undifferentiated, HCW-2 cells induced processing of Bid, substantial efflux of cytochrome c from mitochondria to the cytosol, activation of caspase-3 and apoptosis. The biological significance of the increased mitochondrial Bak in differentiated HCW-2 cells was supported by the finding that transient transfection of a bak cDNA into HCW-2 cells conferred sensitivity to STS-triggered apoptosis, as determined by pro-caspase-3 processing, cytochrome c efflux and DNA fragmentation. Our results suggest that the induction of Bak, upon monocytic differentiation, may be a critical event that regulates the apoptotic sensitivity of differentiated HCW-2 cells. Oncogene (2000) 19, 4108 - 4116

Topoisomerase II Is Required for Mitoxantrone to Signal Nuclear Factor κB Activation in HL60 Cells

Topoisomerase II is a target for a number of chemotherapeutic agents used in the treatment of cancer. Its essential physiological role in modifying the topology of DNA involves the generation of transient double-strand breaks. Anti-cancer drugs, such as mitoxantrone, that target this enzyme interrupt its catalytic cycle and give rise to persistent double strand breaks, which may be lethal to a cell. We investigated the role of such lesions in signaling the activation of the transcription factor nuclear factor kappaB (NFkappaB) by this drug. Mitoxantrone activated NFkappaB and stimulated IkappaBalpha degradation in the promyelocytic leukemia cell line HL60 but not in the variant cells, HL60/MX2 cells, which lack the beta isoform of topoisomerase II and express a truncated alpha isoform that results in an altered subcellular distribution. Treatment of sensitive HL60 cells with mitoxantrone led to a depletion of both isoforms, suggesting the stabilization of transient DNA-topoisomerase II complexes. This depletion was absent in the variant cells, HL60/MX2. Activation of caspase 3 by mitoxantrone was also impaired in the HL60/MX2 cells. NFkappaB activation in response to tumor necrosis factor and bleomycin, the latter causing topoisomerase II-independent DNA damage, was intact in both cell lines. An inhibitor rather than a poison of topoisomerase II, Imperial Cancer Research Fund 187 (ICRF 187) the mechanism of which does not involve the generation of double strand breaks, did not activate NFkappaB, nor did it induce apoptosis in parental HL60 cells. However, ICRF 187 protected against IkappaB degradation in parental HL60 cells in response to mitoxantrone. This protection was also shown with another topoisomerase II inhibitor, merbarone, which is structurally and functionally distinct from ICRF 187. Their effects were specific, as neither protected against tumor necrosis factor-stimulated IkappaB degradation. The poisoning of topoiso- merase II with resultant DNA damage is therefore a critical signal for NFkappaB activation.

Induction of apoptosis by hydrolyzable tannins from Eugeniajambos L. on human leukemia cells

Eugenia jambos L. (Myrtaceae) is an antipyretic and anti-inflammatory herb of Asian folk medicine. A 70% acetone extract exerted the strongest cytotoxic effects on human leukemia cells (HL-60) from a preliminary screening of 15 plants. The cytotoxic principles were separated by bio-assay-guided fractionation to HL-60 cells; two hydrolyzable tannins (1 -O-galloyl castalagin and casuarinin) were isolated from the 70% acetone extract. All significantly inhibited human promyelocytic leukemia cell line HL-60 and showed less cytotoxicity to human adenocarcinoma cell line SK-HEP-1 and normal cell lines of human lymphocytes and Chang liver cells. Thus, these compounds were exhibited the dose-dependent manner in HL-60 cells and the IC 50 were 10.8 and 12.5 μM, respectively. Flow cytometric analysis demonstrated the presence of apoptotic cells with low DNA content, a decrease of cell population at G 2/M phase, and a concomitant increase of cell population at G 1 phase. The apoptosis induced by these two compounds was also demonstrated by DNA fragmentation assay and microscopic observation. These results suggest that the cytotoxic mechanism of both antitumor principle constituents might be the induction of apoptosis in HL-60 cells.

CD44v6 cell surface expression is a common feature of macrophages and macrophage-like cells – implication for a natural macrophage extravasation mechanism mimicked by tumor cells

Soluble CD44standard (sCD44s) and CD44v6 (sCD44v6) cannot only be detected in sera of patients with pancreatic carcinoma but also of healthy blood donors. To investigate whether sCD44s and sCD44v6 are derived from white blood cells, we stimulated whole blood with phytohemagglutinin and interleukin-2, which induced expression of CD44v6 only on monocytes. For further investigations, we used the promyelocytic leukemia cell line Hl-60. Only Hl-60 cells differentiating along the macrophage pathway showed increased expression of CD44s and CD44v6. Furthermore, only macrophages showed increased secretion of sCD44s and sCD44v6. Our data suggest that CD44s and CD44v6 are common adhesion molecules on macrophages and macrophage-like cells.

Role of Growth Hormone Receptor in HL-60 Cell Survival

Although it is presently well established that locally produced growth hormone (GH) plays a major role in the regulation of survival mechanisms in hemopoietic cells, the responsible mechanisms are poorly understood, and the involvement of the GH receptor (GHR) has not even been demonstrated to date. In this work we investigated the presence of GHR in the human promyelocytic leukemia cell line HL-60, as well as the ability of GH treatment to stimulate both GHR and survival signaling pathways downstream GHR. Our results demonstrate that (1) both GHR mRNA and GHR immunoreactivity are present in HL-60 cells; (2) GH treatment results in an increase in the phosphorylation of the GHR-associated Jak2 and Stat3 proteins, indicating the ability of the hormone to induce receptor activation; and (3) activation of GHR increases the activity of Akt, a serine/threonine kinase that plays a prominent role in the regulation of cell survival. Taken together, these results demonstrate that GHR activation promotes survival of HL-60 cells, thus suggesting that GH plays a major role in the regulation of cell survival in the hemopoietic system, via an autocrine/paracrine mechanism.

Myeloperoxidase Is Involved in H2O2-induced Apoptosis of HL-60 Human Leukemia Cells

We examined the mechanism of H(2)O(2)-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H(2)O(2), and at concentrations up to about 20-25 micrometer, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H(2)O(2) do not involve hydroxyl radical. When HL-60 cells were exposed to H(2)O(2) in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN(.) N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H(2)O(2) concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN(.). Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H(2)O(2)-induced apoptosis. Based on this we reasoned that the difference in H(2)O(2)-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H(2)O(2)-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H(2)O(2)-induced apoptosis. Taken together these observations indicate that H(2)O(2)-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN(.).

Cdk inhibitors, roscovitine and olomoucine, synergize with farnesyltransferase inhibitor (FTI) to induce efficient apoptosis of human cancer cell lines.

Farnesyltransferase inhibitor (FTI) induces apoptosis of transformed cells. This involves changes in mitochondria, including decrease of mitochondrial membrane potential and the release of cytochrome c. The released cytochrome c then induces events leading to the activation of caspase-3. In this study, we report that purine derivative cyclin-dependent kinase (Cdk) inhibitors, roscovitine and olomoucine, dramatically enhance this FTI-induced apoptosis of human cancer cell lines. We noticed the synergy between Cdk inhibitors and FTI through our screen to identify compounds that enhance FTI-induced apoptosis of promyelocytic leukemic cell line HL-60. The Cdk inhibitors by themselves do not induce apoptosis at the concentrations used. Roscovitine synergizes with FTI to release cytochrome c from mitochondria. In addition, we detected synergistic effects of FTI and roscovitine to inhibit hyperphosphorylation of retinoblastoma protein. Enhancement of FTI-induced apoptosis by roscovitine is not unique to HL-60 cells, since similar synergy was observed with a leukemic cell line CEM and a prostate cancer cell line LNCaP. In LNCaP cells, in addition to roscovitine and olomoucine, phophatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, was effective in enhancing FTI-induced apoptosis. However, the effects of roscovitine appear to be distinct from those of LY294002, since roscovitine did not affect Akt activity while LY294002 significantly decreased the activity of Akt. Our finding of the synergy between FTI and Cdk inhibitor is significant for understanding the mechanism of action of FTI as well as for clinical use of FTI.

Spicamycin and KRN5500 induce apoptosis in myeloid and lymphoid cell lines with down-regulation of bcl-2 expression and modulation of promyelocytic leukemia protein.

Spicamycin is a potent inducer of differentiation of human myeloid leukemia cells (HL‐60) and murine myeloid leukemia cells (M1). One of the spicamycin derivatives, KRN5500, shows a broad spectrum of antitumor activity against human tumor xenografts in nude mice. In this study, we first investigated the differentiation efficacy of spicamycin and KRN5500 in HL‐60 and acute promyelocytic leukemia cell line, NB4, and found that low concentrations of both compounds induced differentiation to a small extent in both cell lines, but markedly induced apoptosis in NB4 cells. Further investigation in a myeloid leukemia cell line, NKM‐1, a lymphoma cell line, Daudi, and multiple myeloma cell line, NOP‐1, showed that high concentrations of both compounds also induced apoptosis in these cells. The 50% inhibitory concentration (IC50) determined by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay showed that myeloid cells were more sensitive to both compounds than lymphoid cells, and spicamycin was more potent than KRN5500. Western blot analysis of Bcl‐2, Bcl‐xL and Bax expression and immunofluorescence analysis of promyelocytic leukemia (PML) protein indicated that apoptosis induced by spicamycin and KRN5500 was associated with down‐regulation of Bcl‐2 expression and modulation of PML protein. Thus, spicamycin and KRN5500 may be useful for the treatment of myeloid and lymphoid neoplasms.

Plasminogen activator inhibitor 1 may promote tumour growth through inhibition of apoptosis.

Plasminogen activator inhibitor 1 (PAI-1) has been found to be a bad prognostic factor in a number of tumours but the reason has not been fully explained. The human prostate cancer cell line PC-3 and the human promyelocytic leukaemia cell line HL-60 were used in this study to determine the effect of PAI-1 on spontaneous and induced apoptosis in culture. Apoptosis was induced with camptothecin or etoposide. Addition of a stable variant of PAI-1 or wild-type PAI-1 to these cells resulted in a significant inhibition of apoptosis. In contrast, both the latent form of PAI-1 and the stable variant of PAI-1 inactivated by a specific neutralizing monoclonal antibody, or the stable variant of PAI-1 in a complex with recombinant urokinase did not inhibit apoptosis. This indicated that the inhibitory activity of PAI-1 was required for its anti-apoptotic effect but the urokinase-type plasminogen activator receptor was not involved. These findings provide an explanation for the bad prognostic correlation of high PAI-1 levels in tumours. The anti-apoptotic effect was also found in non-tumoural cells including human umbilical vein endothelial cells and the benign human breast epithelial cell line MCF-10A, suggesting that this is a novel physiologic function of PAI-1. © 2000 Cancer Research Campaign

The protein kinase C ?-specific inhibitor LY379196 blocks TPA-induced monocytic differentiation of HL60 cells the protein kinase C ?-specific inhibitor LY379196 blocks TPA-induced monocytic differentiation of HL60 cells

The ability of the promyelocytic leukemia HL60 cell line to differentiate in response to various stimuli has provided a widely used model of differentiation. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), acting via its cellular receptor protein kinase C(PKC), induces these cells to acquire a monocytic phenotype. We set out to identify the specific isoform of the multigene PKC family that is involved in this differentiation event. To do so, we utilized a highly specific PKCbeta inhibitor, LY379196. We found that LY379196 could prevent the growth arrest, cellular adherence, and changes in several marker proteins that occur after the addition of TPA to HL60 cells and that these effects were not simply due to nonspecific cytotoxicity. Thus, the present studies provide strong evidence that the beta isoform of PKC plays a critical role in TPA-induced HL60 monocytic differentiation.