NF-кB inhibition by DHMEQ: in vitro antiproliferative effects on pilocytic astrocytoma and concise review of the current literature.

INTRODUCTION: Pilocytic astrocytoma (PA) is the most common brain tumor that affects the pediatric population. Even though PA is benign and treatment only involves surgery, recurrent or unresectable tumors require chemo- and radiotherapy. Besides BRAF, CDKN2A, or IDH mutations, the hyperactivation of the nuclear factor NF-κB contributes to tumor growth and survival. METHODS: In the present study, we used publicly available data for the in silico analysis of NF-κB subunits (RELA, RELB, REL, NF-κB1, and NF-κB2) expression in PA samples. Besides, in vitro assays were performed to evaluate proliferation, migration, cell death, on the PA cell line Res286 comparing to human primary astrocytes. Sensitization to radiation therapy and temozolomide (TMZ) was also assayed. RESULTS: Our results showed that all the members of the NF-kB family are upregulated in PA datasets compared to normal brain tissues. Moreover, DHMEQ treatment significantly reduced cell growth and motility, while sensitized cells to ionizing radiation and TMZ, as previously seen in high-grade gliomas. CONCLUSIONS: This drug presents a potential application in clinical practice for the treatment of recurrent or inoperable PA. Moreover, its use might assist adjuvant chemotherapy and reduce irradiation doses to avoid toxicity to the surrounding tissues.

Immunosuppression through constitutively activated NF-κB signalling in human ovarian cancer and its reversal by an NF-κB inhibitor.

BACKGROUND: Although T-cell immunity is thought to be involved in the prognosis of epithelial ovarian cancer (EOC) patients, immunosuppressive conditions hamper antitumour immune responses. Thus, their mechanisms and overcoming strategies need to be investigated. METHODS: The role of NF-κB in human EOC cells and macrophages was evaluated by in vitro production of immunosuppressive IL-6 and IL-8 by EOC cells and in vivo analysis of immune responses in nude mice implanted with human EOC cells using an NF-κB inhibitor DHMEQ. RESULTS: In EOC patients, increased plasma IL-6, IL-8, and arginase were observed. The NF-κB inhibitor DHMEQ inhibited the production of IL-6 and IL-8 by EOC cell lines. Immunosuppression of human DCs and macrophages by culture supernatant of EOC cells was reversed with the pretreatment of DHMEQ. Administration of DHMEQ to nude mice implanted with human EOC resulted in the restoration of T-cell stimulatory activity of murine DCs along with the reduction of tumour accumulation and arginase expression of MDSCs. Nuclear factor-κB inhibition in tumour-bearing mice also enhanced antitumour effects of transferred murine naive T cells. CONCLUSIONS: NF-κB is involved in the immunosuppression induced by human EOC, and its inhibitor may restore antitumour immune responses, indicating that NF-κB is an attractive target for EOC treatment.

Enhancement of radiosensitivity by a unique novel NF-κB inhibitor, DHMEQ, in prostate cancer.

BACKGROUND: Inducible activation of nuclear factor (NF)-κB is one of the principal mechanisms through which resistant prostate cancer cells are protected from radiotherapy. We hypothesised that inactivation of inducible NF-κB with a novel NF-κB inhibitor, DHMEQ, would increase the therapeutic effects of radiotherapy. METHODS: PC-3 and LNCaP cells were exposed to irradiation and/or DHMEQ. Cell viability, cell cycle analysis, western blotting assay, and NF-κB activity were measured. The antitumour effect of irradiation combined with DHMEQ in vivo was also assessed. RESULTS: The combination of DHMEQ with irradiation resulted in cell growth inhibition and G2/M arrest relative to treatment with irradiation alone. Inducible NF-κB activity by irradiation was inhibited by DHMEQ treatment. The expression of p53 and p21 in LNCaP, and of 14-3-3σ in PC-3 cells, was increased in the combination treatment. In the in vivo study, 64 days after the start of treatment, tumour size was 85.1%, 77.1%, and 64.7% smaller in the combination treatment group than that of the untreated control, DHMEQ-treated alone, and irradiation alone groups, respectively. CONCLUSION: Blockade of NF-κB activity induced by radiation with DHMEQ could overcome radio-resistant responses and may become a new therapeutic modality for treating prostate cancer.

Dehydroxymethylepoxyquinomicin (DHMEQ), a novel NF-kappaB inhibitor, inhibits allergic inflammation and airway remodelling in murine models of asthma.

BACKGROUND: Dehydroxymethylepoxyquinomicin (DHMEQ) is a newly developed compound that inhibits nuclear factor κB activation and is reported to ameliorate animal models of various inflammatory diseases without significant adverse effects. Because nuclear factor κB is a transcription factor that plays a critical role in the pathophysiology of asthma, DHMEQ may be of therapeutic benefit in asthma. OBJECTIVE: The purpose of this study was to evaluate the effects of DHMEQ on airway inflammation and remodelling in murine models of asthma. METHODS: The BALB/c mice were sensitized and then challenged acutely or chronically with ovalbumin and administered DHMEQ intraperitoneally before each challenge. Inflammation of airways, lung histopathology and airway hyper responsiveness to methacholine challenge were evaluated. In addition, the effect of DHMEQ on production of cytokines and eotaxin-1 by murine splenocytes, human peripheral blood mononuclear cells and bronchial epithelial cells was investigated. RESULTS: Airway hyper responsiveness was ameliorated in both acutely and chronically challenged models by treatment with DHMEQ. DHMEQ significantly reduced eosinophilic airway inflammation and levels of Th2 cytokines in bronchoalveolar lavage fluid in the acute model. It also inhibited parameters of airway remodelling including mucus production, peribronchial fibrosis and the expression of α-smooth muscle actin. Moreover, the production of Th2 cytokines from murine splenocytes and human peripheral blood mononuclear cells and the production of eotaxin-1 by bronchial epithelial cells were inhibited by DHMEQ. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that DHMEQ inhibits allergic airway inflammation and airway remodelling in murine models of asthma. DHMEQ may have therapeutic potential in the treatment of asthma.

Unconventional anisotropic s-wave superconducting gaps of the LiFeAs iron-pnictide superconductor.

We have performed high-resolution angle-resolved photoemission spectroscopy on Fe-based superconductor LiFeAs (T(c)=18 K). We reveal multiple nodeless superconducting (SC) gaps with 2Δ/k(B)T(c) ratios varying from 2.8 to 6.4, depending on the Fermi surface (FS). We also succeeded in directly observing a gap anisotropy along the FS with magnitude up to ~30%. The anisotropy is fourfold symmetric with an antiphase between the hole and electron FSs, suggesting complex anisotropic interactions for the SC pairing. The observed momentum dependence of the SC gap offers an excellent opportunity to investigate the underlying pairing mechanism.

Dehydroxymethylepoxyquinomicin (DHMEQ) can suppress tumour necrosis factor-α production in lipopolysaccharide-injected mice, resulting in rescuing mice from death in vivo.

Dehydroxymethylepoxyquinomicin (DHMEQ), a new nuclear factor (NF)-κB inhibitor, has several beneficial effects, including the suppression of tumour growth and anti-inflammatory effects. DHMEQ can also suppress the production of tumour necrosis factor (TNF)-α induced by lipopolysaccharide (LPS) in vitro. In the present study, we examine the effects of DHMEQ on TNF-α production in vivo and on the survival of mice injected with LPS. When DHMEQ was injected into mice 2 h before LPS injection, the survival of the LPS-injected mice was prolonged. When DHMEQ was injected twice (2 h before LPS injection and the day after LPS injection), all the mice were rescued. The injection of DHMEQ 1 h after LPS injection and the day after LPS injection also resulted in the rescue of all mice. The serum levels of TNF-α in the mice that received both LPS and DHMEQ were suppressed compared to the mice that received only LPS. These results suggest that DHMEQ can be utilized for the prevention and treatment of endotoxin shock.

Gene set enrichment analysis provides insight into novel signalling pathways in breast cancer stem cells.

BACKGROUND: Tumour-initiating cells (TICs) or cancer stem cells can exist as a small population in malignant tissues. The signalling pathways activated in TICs that contribute to tumourigenesis are not fully understood. METHODS: Several breast cancer cell lines were sorted with CD24 and CD44, known markers for enrichment of breast cancer TICs. Tumourigenesis was analysed using sorted cells and total RNA was subjected to gene expression profiling and gene set enrichment analysis (GSEA). RESULTS: We showed that several breast cancer cell lines have a small population of CD24(-/low)/CD44(+) cells in which TICs may be enriched, and confirmed the properties of TICs in a xenograft model. GSEA revealed that CD24(-/low)/CD44(+) cell populations are enriched for genes involved in transforming growth factor-beta, tumour necrosis factor, and interferon response pathways. Moreover, we found the presence of nuclear factor-kappaB (NF-kappaB) activity in CD24(-/low)/CD44(+) cells, which was previously unrecognised. In addition, NF-kappaB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) prevented tumourigenesis of CD24(-/low)/CD44(+) cells in vivo. CONCLUSION: Our findings suggest that signalling pathways identified using GSEA help to identify molecular targets and biomarkers for TIC-like cells.

Rituximab inhibits the constitutively activated PI3K-Akt pathway in B-NHL cell lines: involvement in chemosensitization to drug-induced apoptosis.

Rituximab (chimeric anti-CD20 monoclonal antibody) is currently being used, alone or in combination with chemotherapy, in the treatment of B-non-Hodgkin's lymphoma (B-NHL). We have reported that rituximab treatment of B-NHL cell lines sensitizes the drug-resistant tumor cells to apoptosis by various chemotherapeutic drugs and chemosensitization was, in large part, owing to the selective inhibition of the anti-apoptotic Bcl-(XL) gene product. The constitutive activation of the Akt pathway in B-NHL results in overexpression and functional activation of Bcl-(xL). Hence, we hypothesized that rituximab-induced inhibition of Bcl-(xL) expression and chemosensitization may result, in part, from its inhibitory activity of the Akt pathway. This hypothesis was tested using the drug-resistant Ramos and Daudi B-NHL cell lines. Time kinetic analysis revealed that treatment with rituximab inhibited phosphorylation of Akt, but not unphosphorylated Akt, and the inhibition was first detected at 6 h post-rituximab treatment. Similar time kinetics revealed rituximab-induced inhibition of p-PDK1, p-Bad, p-IKKalpha/beta and p-Ikappabetaalpha and no inhibition of unphosphorylated proteins. In addition, rituximab treatment resulted in significant increase of Bcl-(xL)-Bad heterodimeric complexes as compared to untreated cells. The role of the Akt pathway in the regulation of resistance was corroborated by the use of the Akt inhibitor, LY294002, and by transfection with siRNA Akt. Treatment of tumor cells with LY294002 or with Akt siRNA, but not control siRNA, resulted in inhibition of Bcl-(xL) expression and sensitization to drug-induced apoptosis. Although rituximab did not inhibit the Akt pathway nor sensitized the rituximab-resistant Ramos RR1 clone, treatment with LY294002 or Akt siRNA sensitized the clone to drug-induced apoptosis. The present findings demonstrate for the first time that rituximab inhibits the constitutively activated Akt pathway in B-NHL cell lines, and this inhibition contributes to sensitization of drug-resistant cells to apoptosis by chemotherapeutic drugs. The findings also identify the Akt pathway as target for therapeutic intervention in the reversal of rituximab and drug-resistant B-NHL.

Nitric oxide mediates murine cytomegalovirus-associated pneumonitis in lungs that are free of the virus.

4 wk after intraperitoneal inoculation of 0.2 LD50 (50% lethal dose) of murine cytomegalovirus (MCMV) in adult BALB/c mice, MCMV remained detectable in the salivary glands, but not in the lungs or other organs. When the T cells of these mice were activated in vivo by a single injection of anti-CD3 monoclonal antibody, interstitial pneumonitis was induced in the lungs that were free of the virus with an excessive production of the cytokines. In the lungs of such mice persistently infected with MCMV, the mRNA of the cytokines such as IL-2, IL-6, TNF-alpha, and IFN-gamma were abundantly expressed 3 h after the anti-CD3 injection, and the elevated levels continued thereafter. A marked expression of inducible nitric oxide synthetase (iNOS) was then noted in the lungs, suggesting that such cytokines as TNF-alpha and IFN-gamma may have induced iNOS. Although the increase in NO formation was demonstrated by the significant elevation of the serum levels of nitrite and nitrate, the interstitial pneumonitis was not associated with either increased superoxide formation or peroxynitrite-induced tyrosine nitration. Nevertheless, the administration of an NO antagonist also alleviated the interstitial pneumonitis provoked by anti-CD3 mAb. Based on these findings, it was concluded that MCMV-associated pneumonitis is mediated by a molecule of cytokine-induced NO other than peroxynitrite.

DHMEQ, a new NF-kappaB inhibitor, induces apoptosis and enhances fludarabine effects on chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is a low-grade lymphoid malignancy incurable with conventional modalities of chemotherapy. Strong and constitutive nuclear factor kappa B (NF-kappaB) activation is a characteristic of CLL cells. We examined the effects of a new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on CLL cells. Dehydroxymethylepoxyquinomicin completely abrogated constitutive NF-kappaB activity and induced apoptosis of CLL cells. Apoptosis induced by DHMEQ was accompanied by downregulation of NF-kappaB-dependent antiapoptotic genes: c-IAP, Bfl-1, Bcl-X(L) and c-FLIP. Dehydroxymethylepoxyquinomicin also inhibited NF-kappaB induced by CD40 and enhanced fludarabine-mediated apoptosis of CLL cells. Results of this study suggest that inhibition of constitutive and inducible NF-kappaB by DHMEQ in combination with fludarabine is a promising strategy for the treatment of CLL.

Thrombomodulin blocks calcineurin inhibitor-induced vascular permeability via inhibition of Src/VE-cadherin axis.

Recombinant human soluble thrombomodulin (rTM) counteracted capillary leakage and alleviated edema in individuals with sinusoidal obstruction syndrome and engraftment syndrome after hematopoietic stem cell transplantation. We previously showed that rTM increased levels of antiapoptotic protein Mcl-1 and protected endothelial cells from calcineurin inhibitor cyclosporine A (CsA)-induced apoptosis. However, the molecular mechanisms by which rTM enhances barrier function in vascular endothelial cells remain unknown. Here we show that exposure of vascular endothelial EA.hy926 cells to CsA induced phosphorylation of Src/vascular endothelial cadherin (VE-cadherin) and translocation of VE-cadherin from cell surface to cytoplasm, resulting in an increase in vascular permeability. In addition, CsA increased production of inflammatory cytokines, including interleukin (IL)-1ß and IL-6, associated with an increase in nuclear levels of nuclear factor-κB (NF-κB) which also enhanced vascular permeability. Importantly, the fourth and fifth regions of epidermal growth factor-like domain of TM (TME45) attenuated CsA-induced p-Src/VE-cadherin and vascular permeability in parallel with a decrease in nuclear levels of NF-κB and cytokine production in EA.hy926 cells. In conclusion, TM, especially TME45, maintains vascular integrity, at least in part, via Src signaling.

Inhibition of macrophage activation and phagocytosis by a novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin.

Previously, we designed and synthesized a new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ). In the present research we looked into the effect of DHMEQ on the activation of macrophages, especially on the phagocytotic activity of cells of the mouse macrophage-like cell line RAW264.7. DHMEQ inhibited lipopolysaccharide (LPS)-induced NF-kappaB activation by inhibiting its nuclear translocation from the cytoplasm. It also inhibited the expression of inducible NO synthase (iNOS) and nitric oxide (NO) production induced by LPS and interferon-gamma. Using enzyme-linked immunosorbent assays (ELISAs) we showed DHMEQ to inhibit LPS-induced secretion of IL-6, IL-12, interleukin-1beta (IL-1beta), and TNF-alpha. Furthermore, DHMEQ also inhibited the phagocytosis of fluorescently labeled Escherichia coli by RAW264.7 cells treated with LPS or IL-1beta, thus being the first evidence for the involvement of NF-kappaB in the regulation of phagocytosis by use of this inhibitor. Deletion of p65 by siRNA also inhibited the phagocytosis. DHMEQ inhibited the LPS-induced but not IL-1beta-induced phagocytosis of glass beads, indicating that activation of not only NF-kappaB but also Toll-like receptor 4 (TLR-4) is essential for the phagocytosis of E. coli. Previously we found that DHMEQ inhibited type 2 collagen-induced rheumatoid arthritis and the growth of various human carcinomas in mice. It is thus likely that inhibition of macrophage activation is involved in the mechanism of these anti-inflammatory and antitumor activities of DHMEQ in mice.

Mutagenicity of aclacinomycin A and daunomycin derivatives.

An antitumor antibiotic aclacinomycin A, was nonmutagenic in a Salmonella test, but its derivative, N-demethylaclacinomycin A, was mutagenic. Similarly, 1-deoxypyrromycin, a hydrolysis product of aclacinomycin A, was nonmutagenic, but N-demethyl-1-deoxypyrromycin was mutagenic. Daunomycin was highly mutagenic, but N-methyldaunomycin showed only weak mutagenicity, and N-dimethyldaunomycin was nonmutagenic. The aglycones of aclacinomycin A and daunomycin were not mutagenic. Thus, the amino moiety of anthracycline glycosides is concluded to be essential for mutagenesis.

Inhibition of experimental metastasis by an alpha-glucosidase inhibitor, 1,6-epi-cyclophellitol.

Isolated from a culture filtrate of Phellinus sp., cyclophellitol is a specific inhibitor of beta-glucosidase, but unlike castanospermine, it does not inhibit experimental metastasis. However, its structural analogue, 1,6-epi-cyclophellitol, inhibited alpha-glucosidase as well as beta-glucosidase, and inhibited experimental metastasis. 1,6-Epi-cyclophellitol depressed alpha-glucosidase activity in cultured B16/F10 cells after 48 h of incubation. Preincubation of B16/F10 cells for 48 h with 1,6-epi-cyclophellitol inhibited invasion of the cells in a Boyden chamber assay at the doses effective in inhibiting alpha-glucosidase in situ. Pulmonary metastasis of B16/F10 cells in mice was inhibited by pretreatment of the cells with 1,6-epi-cyclophellitol in culture. The inhibitor reduced the collagen type I- and IV-mediated attachment of the cells, whereas it had no effect on laminin-mediated attachment. These results suggest that alpha-glucosidase in tumor cells is essential for the metastatic process through the cellular interaction with collagen type I and IV.

Induction by the guanosine analogue oxanosine of reversion toward the normal phenotype of K-ras-transformed rat kidney cells.

Oxanosine, a guanosine analogue antibiotic, altered the "transformed" morphology of rat kidney cells integrating a temperature-sensitive K-ras gene into "normal" morphology at 34 degrees C, a permissive temperature. Oxanosine also increased the cellular content of fibronectin to the normal level. The cells under these conditions had lower levels of guanine nucleotides and unstable and less palmitylated Mr 21,000 protein (K-ras gene product, referred to as p21). These changes in p21 were the same as those which occurred to p21 in the cells cultured in the absence of oxanosine at 39.5 degrees C, a nonpermissive temperature.

Involvement of hydrogen peroxide production in erbstatin-induced apoptosis in human small cell lung carcinoma cells.

Tyrosine kinase inhibitor, erbstatin, induced morphological apoptosis and DNA fragmentation in human small cell lung carcinoma (SCLC) cells. Erbstatin-induced apoptosis was inhibited by antioxidants, whereas erbstatin-inhibited tyrosine phosphorylation was not affected by them. Erbstatin was shown by means of flow cytometry to induce hydrogen peroxide generation. Furthermore, hydrogen peroxide induced morphological apoptosis and DNA fragmentation in the SCLC cells. We also demonstrated that erbstatin-induced hydrogen peroxide production and DNA fragmentation were partially suppressed by inhibition of protein synthesis. Thus, erbstatin-induced apoptosis would be due to hydrogen peroxide generation via newly synthesized protein.

Inhibition of experimental blood-borne lung metastasis by protease inhibitors.

The inhibitory effects of protease inhibitors on blood-borne metastasis in male Donryu rat lung were studied. Injection i.v. of 10(6) Yoshida ascites hepatoma AH7974 cells induced about 118 +/- 92 (S.D.) metastatic foci in rat lung after 3 weeks. Leupeptin (50 mg/kg body weight twice a day), injected i.p. from 2 days before to 4 days after the inoculation of tumor cells, reduced the number of metastatic foci to about 49 +/- 45 (p less than 0.005). Leupeptin also suppressed the formation of metastatic foci of Yoshida ascites hepatoma AH100B cells (p less than 0.001). Elastatinal (100 mg/kg body weight twice a day) and chymostatin (100 mg/kg body weight once a day) did not inhibit formation of metastatic foci of AH7974 cells. Injection i.v. of 10(6) AH7974 cells induced pulmonary thrombi within 1 hr. Leupeptin (50 mg/kg body weight twice a day) reduced the number of thrombi from 1298 +/- 395 to 646 +/- 218, when injected i.p. for 2 days before the inoculation of the cells (p less than 0.005). Chymostatin and elastatinal did not significantly change the number of pulmonary thrombi. These results indicate that leupeptin inhibited metastasis formation and suggest that this effect may be due to the inhibition of thrombus formation after the arrest of circulating tumor cells.

Neuronal apoptosis by apolipoprotein E4 through low-density lipoprotein receptor-related protein and heterotrimeric GTPases.

The epsilon4 genotype of apolipoprotein E (apoE4) is the most established predisposing factor in Alzheimer's disease (AD); however, it remains unclear how apoE4 contributes to the pathophysiology. Here, we report that the apoE4 protein (ApoE4) evokes apoptosis in neuronal cells through the low-density lipoprotein receptor-related protein (LRP) and heterotrimeric GTPases. We examined neuron/neuroblastoma hybrid F11 cells and found that these cells were killed by 30 microg/ml ApoE4, but not by 30 microg/ml ApoE3. ApoE4-induced death occurred with typical features for apoptosis in time- and dose-dependent manners, and was observed in SH-SY5Y neuroblastomas, but not in glioblastomas or non-neuronal Chinese hamster ovary cells. Activated, but not native, alpha2-macroglobulin suppressed this ApoE4 toxicity. Suppression by the antisense oligonucleotide to LRP and inhibition by low nanomolar concentrations of LRP-associated protein RAP provided evidence for the involvement of LRP. The involvement of heterotrimeric GTPases was demonstrated by the findings that (1) ApoE4-induced death was suppressed by pertussis toxin (PTX), but not by heat-inactivated PTX; and (2) transfection with PTX-resistant mutant cDNAs of Galpha(i) restored the toxicity of ApoE4 restricted by PTX. We thus conclude that one of the neurotoxic mechanisms triggered by ApoE4 is to activate a cell type-specific apoptogenic program involving LRP and the G(i) class of GTPases and that the apoE4 gene may play a direct role in the pathogenesis of AD and other forms of dementia.

Involvement of tyrosine kinase in growth factor-induced phospholipase C activation in NIH3T3 cells.

Tyrosine kinase inhibitors such as erbstatin and lavendustin derivative inhibited platelet-derived growth factor (PDGF)- and bombesin-induced inositol phosphate formation and phospholipase C (PLC) activation in quiescent NIH3T3 cells. However, bombesin-induced PLC activation was only partially inhibited by tyrosine kinase inhibitors, whereas PDGF-induced activation was completely. Moreover, although bombesin-induced PLC activation was partially inhibited by pertussis toxin alone, this toxin inhibited almost completely in the presence of tyrosine kinase inhibitors. Thus, tyrosine kinase was suggested to be involved in PDGF- and bombesin-induced PLC activation in a different manner.

Activation of the respiratory burst and tyrosine phosphorylation of proteins in human neutrophils: no direct relationship and involvement of protein kinase C-dependent and -independent signaling pathways.

Human neutrophils maximally stimulated with the optimal concentration (100 ng/ml) of phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), for 5 min at 37 degrees C did not respond with superoxide (O2-) release to the later addition of PMA itself or the Ca2+ ionophore ionomycin. However, these cells did respond with enhanced release of O2- to the later addition of N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A (Con A). In these PMA-pretreated cells, an increase in cytoplasmic free Ca2+ ([Ca2+]i) induced by ionomycin was unaffected, whereas that induced by FMLP was inhibited by 50-60% and that induced by Con A was completely abolished. A 42-kDa protein was predominantly and consistently tyrosine-phosphorylated by FMLP, PMA and ionomycin with the different kinetics according to the stimuli. The dose-response curves showed that tyrosine phosphorylation and O2- release were stimulated in parallel by PMA, whereas tyrosine phosphorylation and an increase in [Ca2+]i, but not O2- release, were stimulated in parallel by FMLP or ionomycin. The potency of inducing tyrosine phosphorylation was ionomycin > FMLP = PMA, whereas the potency of triggering of O2- release was PMA > ionomycin = FMLP. UCN-01, a PKC inhibitor, inhibited O2- release and tyrosine phosphorylation induced by PMA, but not by FMLP or ionomycin. In contrast, pertussis toxin inhibited O2- release and tyrosine phosphorylation induced by FMLP, but not by PMA. Tyrosine kinase inhibitors (erbstatin and genistein) inhibited O2- release induced by FMLP, but not by PMA. However, both tyrosine kinase inhibitors did not impair FMLP- or PMA-induced tyrosine phosphorylation of a 42-kDa protein. Increased tyrosine phosphorylation of a 42-kDa protein was also detected in immature myeloid cells (HL-60 cells) stimulated by PMA, but not by ionomycin. These findings suggest that FMLP and Con A trigger the respiratory burst in human neutrophils by activating the definite pathway which include other signals than activation of PKC and an increase in [Ca2+]i; tyrosine phosphorylation of a 42-kDa protein is induced by the PKC-dependent and independent mechanisms according to the stimuli, and the PKC-independent and ionomycin-sensitive mechanism is inoperative in HL-60 cells; and tyrosine phosphorylation of a 42-kDa protein is unlikely to be causally related to activation of the respiratory burst.