Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells.

TGF-beta 1 controls the expression of numerous genes, including early response and cellular matrix genes. However, the signal-transducing mechanism underlying this regulation of gene expression is not fully understood. In this study, we investigated whether redox regulation plays a role in the TGF-beta 1 signal transduction in the mouse osteoblastic cell line (MC3T3-E1). The overall intracellular oxidized state of the cells, when measured using 2',7'-dichlorofluorescin diacetate by laser-scanning confocal microscopy, was increased transiently after the addition of TGF-beta 1. This increase was abolished by the addition of oxygen radical scavengers such as catalase and N-acetylcysteine. In a variant cell line lacking the TGF-beta 1 receptor, the intracellular oxidized state was not modulated by treatment with TGF-beta 1. We then examined the expression of early growth response-1 (egr-1) gene, which is inducible by TGF-beta 1 and H2O2. Radical scavengers inhibited the induction of egr-1 by TGF-beta 1, but not that by 12-O-tetradecanoylphorbol-13 acetate. A nuclear run-on assay indicated that this inhibition was at the transcriptional level. From transient expression experiments using chloramphenicol acetyltransferase gene linked to serially deleted egr-1 gene 5'-upstream region, the CArG element in the 5' flanking region of egr-1 was identified as an essential sequence in the transcriptional activation for both TGF-beta 1 and H2O2 stimulation. These findings suggest that H2O2 acts as a mediator for the TGF-beta 1-induced transcription of egr-1 gene.

Characterization of Jumping translocation breakpoint (JTB) gene product isolated as a TGF-beta1-inducible clone involved in regulation of mitochondrial function, cell growth and cell death.

Jumping translocation breakpoint (JTB) is a gene located on human chromosome 1 at q21 that suffers an unbalanced translocation in various types of cancers, and potentially encodes a transmembrane protein of unknown function. The results of cancer profiling indicated that its expression was suppressed in many cancers from different organs, implying a role in the neoplastic transformation of cells. Recently, we isolated JTB as a TGF-beta1-inducible clone by differential screening. In this study, we characterized its product and biological functions. We found that it was processed at the N-terminus and located mostly in mitochondria. When expressed in cells, JTB-induced clustering of mitochondria around the nuclear periphery and swelling of each mitochondrion. In those mitochondria, membrane potential, as monitored with a JC-1 probe, was significantly reduced. Coinciding with these changes in mitochondria, JTB retarded the growth of the cells and conferred resistance to TGF-beta1-induced apoptosis. These activities were dependent on the N-terminal processing and induced by wild-type JTB but not by a mutant resistant to cleavage. These findings raised the possibility that aberration of JTB in structure or expression induced neoplastic changes in cells through dysfunction of mitochondria leading to deregulated cell growth and/or death.

Hic-5-reduced cell spreading on fibronectin: competitive effects between paxillin and Hic-5 through interaction with focal adhesion kinase.

Hic-5 is a paxillin homologue that is localized to focal adhesion complexes. Hic-5 and paxillin share structural homology and interacting factors such as focal adhesion kinase (FAK), Pyk2/CAKbeta/RAFTK, and PTP-PEST. Here, we showed that Hic-5 inhibits integrin-mediated cell spreading on fibronectin in a competitive manner with paxillin in NIH 3T3 cells. The overexpression of Hic-5 sequestered FAK from paxillin, reduced tyrosine phosphorylation of paxillin and FAK, and prevented paxillin-Crk complex formation. In addition, Hic-5-mediated inhibition of spreading was not observed in mouse embryo fibroblasts (MEFs) derived from FAK(-/-) mice. The activity of c-Src following fibronectin stimulation was decreased by about 30% in Hic-5-expressing cells, and the effect of Hic-5 was restored by the overexpression of FAK and the constitutively active forms of Rho-family GTPases, Rac1 V12 and Cdc42 V12, but not RhoA V14. These observations suggested that Hic-5 inhibits cell spreading through competition with paxillin for FAK and subsequent prevention of downstream signal transduction. Moreover, expression of antisense Hic-5 increased spreading in primary MEFs. These results suggested that the counterbalance of paxillin and Hic-5 expression may be a novel mechanism regulating integrin-mediated signal transduction.

Induction of senescence-like phenotypes by forced expression of hic-5, which encodes a novel LIM motif protein, in immortalized human fibroblasts.

The hic-5 gene encodes a novel protein with Zn finger-like (LIM) motifs, the expression of which increases during cellular senescence. The ectopic expression of hic-5 in nontumorigenic immortalized human fibroblasts, whose expression levels of hic-5 were significantly reduced in comparison with those of mortal cells, decreased colony-forming efficiency. Stable clones expressing high levels of hic-5 mRNA showed higher levels of mRNAs for several extracellular matrix-related proteins, along with the alteration of an alternative splicing as seen in senescent cells and decreased c-fos inducibility. Furthermore, these clones acquired a senescence-like phenotype, such as growth retardation; senescence-like morphology; and increased expression of Cip1/WAF1/sdi1 after 20 to 40 population doublings. On the other hand, antisense RNA expression of hic-5 in human normal diploid fibroblasts delayed the senescence process. HIC-5 was localized in nuclei and had affinity for DNA. Based on these observations, we speculated that HIC-5 affected the expression of senescence-related genes through interacting with DNA and thereby induced the senescence-like phenotypes. To our knowledge, hic-5 is the first single gene that could induce senescence-like phenotypes in a certain type of immortalized human cell and mediate the normal process of senescence.

Stimulation by hydrogen peroxide of DNA synthesis, competence family gene expression and phosphorylation of a specific protein in quiescent Balb/3T3 cells.

Treatment of quiescent Balb/3T3 clone A31-1-1 cells with 0.1-0.2 mM H2O2 in the presence of 1 microM insulin induced DNA synthesis 20-24 h later at almost the same level as that in cells treated with 10% serum. Treatment with 0.1-0.2 mM H2O2 alone did not induce DNA synthesis and was not toxic to the cells. Cell cycle analysis indicated that treatment with H2O2 plus insulin induced progression of the cell cycle from the quiescent state. The amounts of mRNA for competence family genes such as c-fos, KC and JE were increased by the addition of H2O2. Under these conditions H2O2 caused rapid phosphorylation of a protein of 78 kDa with a pI of 6.3 (p78). Phosphorylation of p78 increased on treatment with TPA and serum as well. Catalase reduced the increase in phosphorylation of p78 induced by TPA and serum. Endogenous production of H2O2 was observed within 10 min after treatment of quiescent cells with platelet derived growth factor (PDGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). These results indicate that H2O2 at certain concentrations mimics the action of competence factors on resting Balb/3T3 cells.

Loss of anchorage primarily induces non-apoptotic cell death in a human mammary epithelial cell line under atypical focal adhesion kinase signaling.

Anchorage dependence of cellular growth and survival prevents inappropriate cell growth or survival in ectopic environments, and serves as a potential barrier to metastasis of cancer cells. Therefore, obtaining a better understanding of anchorage-dependent responses in normal cells is the first step to understand and impede anchorage independence of growth and survival in cancer cells and finally to eradicate cancer cells during metastasis. Anoikis, a type of apoptosis specifically induced by lack of appropriate cell-extracellular matrix adhesion, has been established as the dominant response of normal epithelial cells to anchorage loss. For example, under detached conditions, the untransformed mammary epithelial cell (MEC) line MCF-10 A, which exhibits myoepithelial characteristics, underwent anoikis dependent on classical ERK signaling. On the other hand, recent studies have revealed a variety of phenotypes resulting in cell death modalities distinct from anoikis, such as autophagy, necrosis, and cornification, in detached epithelial cells. In the present study, we characterized detachment-induced cell death (DICD) in primary human MECs immortalized with hTERT ((Tert)HMECs), which are bipotent progenitor-like cells with a differentiating phenotype to luminal cells. In contrast to MCF-10 A cells, apoptosis was not observed in detached (Tert)HMECs; instead, non-apoptotic cell death marked by features of entosis, cornification, and necrosis was observed along with downregulation of focal adhesion kinase (FAK) signaling. Cell death was overcome by anchorage-independent activities of FAK but not PI3K/AKT, SRC, and MEK/ERK, suggesting critical roles of atypical FAK signaling pathways in the regulation of non-apoptotic cell death. Further analysis revealed an important role of TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand) as a mediator of FAK signaling in regulation of entosis and necrosis and a role of p38 MAPK in the induction of necrosis. Overall, the present study highlighted outstanding cell subtype or differentiation stage specificity in cell death phenotypes induced upon anchorage loss in human MECs.

Forced expression of hic-5, a senescence-related gene, potentiates a differentiation process of RCT-1 cells induced by retinoic acid.

The hic-5 gene encodes the novel LIM protein which has been implicated in cellular senescence and differentiation processes. Previous studies of rat calvarial cells stimulated to differentiate by addition of retinoic acid (R.A.) showed a four-fold increase in hic-5 expression which preceded an increase in the expression of the differentiation markers, alkaline phosphatase and alpha (I) pro-collagen mRNA. These data suggest involvement of hic-5 in osteoblast differentiation. This hypothesis was further examined using rat calvarial RCT-1 cells containing expression vectors of hic-5. The over-expressing clones showed a decrease in proliferation and displayed the differentiation-related phenotypes such as increased basal levels of alpha (I) collagen mRNA expression and high inducibility by R.A. of alkaline phosphatase activity. Conversely, introduction of hic-5 anti-sense vector leads to the inhibition of alpha (I) collagen mRNA following induction by R.A. These results suggest that hic-5 is one of the regulatory molecules involved in the R.A. induced differentiation process of RCT-1 cells.

Genomic structure and chromosomal mapping of the mouse hic-5 gene that encodes a focal adhesion protein.

The hic-5 gene encodes a focal adhesion protein that has striking similarity to paxillin. Genomic clones of the mouse hic-5 gene were isolated, and included 10 exons that covered the whole mouse mRNA sequence. Comparison of the sequence with those in the expressed sequence tag database suggested that the hic-5 gene contained an extra exon (named exon 1') located about 1kb upstream of exon 1, and mouse cells seemed to express two alternatively spliced forms of mRNA. All the exon-intron boundaries followed the GT/AG rule. Physical mapping and fluorescent in situ hybridization analysis indicated that the hic-5 gene is located on mouse chromosome 7, 60. 0cM from the centromere.

Inhibition by N-acetyl-L-cysteine of interleukin-6 mRNA induction and activation of NF kappa B by tumor necrosis factor alpha in a mouse fibroblastic cell line, Balb/3T3.

Redox-based modulation plays a role in transcriptional control of gene expression. In the present study, we investigated the possible role of reactive oxygen species in the induction of interleukin-6 (IL-6) mRNA and in increases in NF kappa B binding activity by tumor necrosis factor (TNF) alpha using a mouse fibroblastic cell line, Balb/3T3. Expression of IL-6 mRNA is known to be dependent upon NF kappa B that binds to the 5'-flanking region of the IL-6 gene. We found that: (i) TNF alpha increased IL-6 mRNA levels and this increase was inhibited by N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species. (ii) NF kappa B binding activity in this cell line was also increased by TNF alpha, and the increase was inhibited in the presence of NAC. (iii) The treatment of cells with low doses of hydrogen peroxide increased the NF kappa B binding activity. (iv) Expression of a reporter gene in which the chloramphenicol acetyltransferase (CAT) gene was under the control of NF kappa B binding sites was induced by hydrogen peroxide. These results suggest that the induction of IL-6 mRNA is regulated by a mechanism involving reactive oxygen species and that NF kappa B, whose activity is sensitive to the cellular redox state, plays an important role in this induction in a fibroblastic cell line, Balb/3T3, stimulated with TNF alpha.

Isolation of a novel ras-recision gene that is induced by hydrogen peroxide from a mouse osteoblastic cell line, MC3T3-E1.

Hydrogen peroxide appears to mediate growth factor actions, and it inhibits DNA synthesis in normal mouse osteoblastic cells (MC3T3-E1) at non-toxic doses. However the sensitivity of cells to H2O2 is greatly decreased in their ras-transformants. To understand the molecular basis of this sensitivity to H2O2, we attempted to identify H2O2-inducible cDNA clones from MC3T3 cells by differential screening of cDNA libraries, and one of such genes, named HIC-53, was isolated. The level of HIC-53 mRNA was moderately increased by H2O2 as well as by calcium ionophore or dexamethasone, but was not increased by the addition of serum, tumor promoting phorbol ester, or epidermal growth factor. Among mouse organs, HIC-53 mRNA levels were higher in the kidney and lung, but were almost undetectable in the brain, heart, bone, muscle or spleen. In MC3T3 cells transformed with v-Ki-ras, the HIC-53 mRNA level was markedly decreased, and effect of H2O2 was abolished. Although the biological function of HIC-53 is unknown at present, the predicted amino acid sequence exhibited some similarity with bovine cardiac Na+/Ca+ exchanger. The nucleotide sequence of HIC-53 cDNA showed no significant similarity with other known gene sequences.

Pituitary adenylate cyclase-activating polypeptide receptors during development: expression in the rat embryo at primitive streak stage.

The distribution and localization of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor the PAC1 receptor (previously called the type 1 PACAP receptor or PVR1), which binds PACAP, but not vasoactive intestinal peptide, with high affinity] were first investigated in rats with in situ hybridization for its messenger RNA, and with immunohistochemical methods during prenatal and postnatal development. The expression of PACAP receptor messenger RNA was first detected in the rat embryo at the primitive streak stage as early as embryonic day 9, and it was intensely expressed in the neural plate. PACAP receptor messenger RNA was also intensely expressed in the neuroepithelia of the mesencephalon and rhombencephalon at embryonic day 11, and expressed in the basal telencephalon, hippocampal formation neuroepithelium, cortical neuroepithelium and cerebellar neuroepithelium after embryonic day 13. It was also expressed in the olfactory bulb neuroepithelium after embryonic day 16, and in mature regions of the older embryos. In postnatal developing brains, PACAP receptor messenger RNA was intensely expressed in the olfactory bulb, hippocampal formation, cerebellum and other scattered regions. The localization of PACAP receptor-like immunoreactivity coincided well with that of the gene transcripts. We also used reverse transcription-polymerase chain reaction methods to determine the expression of the splice variants of the PACAP receptor gene. At each ontogenetic stage of the rat from embryonic day 9 to postnatal day 60, two major products were detected with reverse transcription-polymerase chain reaction, a thick band (303 base pairs) corresponding to the short splice variant of the receptor that lacks both the "hip" and "hop" cassettes, and a thin band (387 base pairs) corresponding to the splice variant that contains one cassette of "hop" or "hip". There was no evidence for the other larger splice variants. Some of the amplified products were sequenced and found to have the exact sequences of "PACAP receptor" and "PACAP receptor-hopl", which are coupled to different signal transduction pathways. These results indicate that the PACAP receptor is actively expressed in different neuroepithelia from early developmental stages and expressed in various brain regions during prenatal and postnatal development, and that the major splice variants are "PACAP receptor" and "PACAP receptor-hopl". The initial mapping of ontogenetic localization of the PACAP receptor provides the basis for a better understanding of the functions of PACAP and its receptors during the development of the brain.

Cellular distribution of the splice variants of the receptor for pituitary adenylate cyclase-activating polypeptide (PAC(1)-R) in the rat brain by in situ RT-PCR.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide and its specific receptor (the PAC(1) receptor) is widely distributed in the rat brain. It has been reported that alternative splicing of the region encoding the third intracellular loop of the PAC(1) receptor generates six isoforms which are differentially coupled to signal transduction pathways, but the precise distribution and localization of these splice isoforms in the brain remain to be determined. Using the initial specific primer pairs which correspond to the 'hip' or 'hop' types of receptors for the solution-phase reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated that the major splice variants of the PAC(1) receptor in various regions of the rat brain are the short splice isoform 'PAC(1)-R-s' which does not contain either the 'hip' or 'hop' cassette and the another splice isoform, 'PAC(1)-R-hop', which contains the 'hop' cassette. With an innovative molecular histochemical technique, in situ RT-PCR, we determined that these two splice isoforms are both intensely expressed in the mitral cells of the olfactory bulb, the Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and neocortex, and many neurons in the nuclei of hypothalamus and thalamus as well as other regions. The initial mapping of the cell type-specific expression of these two splice variants of the PAC(1) receptor provides the basis for a better understanding of the functional significance of the PAC(1)-R and its ligand PACAP in various brain regions.

Transcriptional induction of MMP-10 by TGF-beta, mediated by activation of MEF2A and downregulation of class IIa HDACs.

Transforming growth factor (TGF)-beta regulates the expression of matrix metalloproteinases (MMPs) and components of the extracellular matrix, thereby profoundly affecting the microenvironment of cells including cancerous ones. We studied MMP-10 induction by TGF-beta in mammary epithelial cells and found that the induction was dependent on the myocyte enhancer factor (MEF)-2 transcription factor. TGF-beta upregulated the gene promoter through the MEF2 site, and knockdown of the MEF2A transcription factor negatively affected MMP-10 induction, whereas its overexpression had a positive effect on the induction. In response to TGF-beta, acetylation and concomitant binding of MEF2A to the promoter region increased, thus suggesting a critical role of MEF2A in transactivation of MMP-10 by TGF-beta. Consistent with the fact that class IIa histone deacetylases (HDACs) interact with MEF2 and suppress transcription, knockdown of HDACs increased and their overexpression inhibited MMP-10 expression. Intriguingly, TGF-beta promoted proteasome-dependent degradation of HDACs. Consistent with this, acetylation of core histones was increased around the MEF2 site of the MMP-10 promoter by TGF-beta and alleviated by overexpression of HDACs. Collectively, it is possible that TGF-beta transcriptionally upregulated MMP-10 through activation of MEF2A, concomitant with acetylation of core histones increasing around the promoter, as a consequence of degradation of the class IIa HDACs.

Significance of nuclear relocalization of ERK1/2 in reactivation of c-fos transcription and DNA synthesis in senescent fibroblasts.

Two of mitogen-activated protein kinases (MAPK), p44(mapk)/p42(mapk) extracellular signal-regulated kinases (ERK1/2), translocate into nuclei following activation and play critical roles in connecting the signal to gene expression and allowing cell-cycle entry. Here we found that the nuclear translocation of ERK1/2 in response to growth stimuli was significantly inhibited in senescent cells that were irreversibly growth arrested, compared with presenescent cells. The activation step of these enzymes was not impaired, since ERK1/2 were phosphorylated and activated in senescent cells as efficiently as in presenescent cells. By elaborately localizing ERK2 in the nuclei of senescent cells, we could restore c-fos transcriptional activity upon growth stimuli, which was repressed in senescent cells. Furthermore, the nuclear localization of ERK1/2 has been suggested to potentiate the proliferative activity of the senescent cells in collaboration with adenovirus E1A protein. More importantly, SV40 large T antigen, the strong inducer of DNA synthesis, had the inherent ability to restore nuclear relocalization of active ERK1/2 in senescent cells, which was essentially required for the reinitiation of DNA synthesis. Thus, manipulating the relocalization of ERK1/2 into nuclei was expected to open the way to overcome some of the senescent phenotypes.

Induction of early response genes by hypergravity in cultured mouse osteoblastic cells (MC3T3-E1).

Hypergravity as low as 50g transiently stimulated cultured mouse osteoblastic cells (MC3T3-E1) to induce early response genes such as c-fos and egr-1, whereas expression of c-jun was marginally affected. The maximum induction of c-fos required more than 90g, but egr-1 induction became maximum below 50g. Staurosporin inhibited the induction of c-fos by hypergravity almost completely at a concentration of 0.1 microM, but it inhibited the induction of egr-1 only partially. In cells pretreated with 12-O-tetradecanoylphorbol 13-acetate, induction of c-fos by hypergravity was almost completely abolished, whereas that of egr-1 was not affected. Activity of protein kinase C seemed to be activated in cells centrifuged at 900g. These results indicate that hypergravity stimulates multiple signal transduction cascades that are connected with the expression of early response genes.

Superoxide as a signal for increase in intracellular pH.

We examined the role of superoxide in the increase in intracellular pH (pHi) of human histiocytic leukemia U937 cells treated with 4 beta-phorbol-12,13-didecanoate (4 beta-PDD) or serum. 4 beta-PDD or serum induced a rapid increase in pHi, and antioxidants such as superoxide dismutase (SOD), vitamin E, and butylated hydroxyanisole (BHA) were found to inhibit the amiloride-sensitive increase in pHi induced by 4 beta-PDD. SOD inhibited the increase in pHi caused by serum, and essentially the same was found in concanavalin A-stimulated mouse thymocytes. Also, a superoxide-generating system, xanthine-xanthine oxidase (X-XOD), increased pHi of U937 cells as much as 4 beta-PDD or serum. From these findings, it appears that superoxide is the basis for the modulation of pHi.

Cell-cycle dependent phosphorylation of HSP28 by TGF beta 1 and H2O2 in normal mouse osteoblastic cells (MC3T3-E1), but not in their ras-transformants.

Transforming growth factor (TGF) beta 1 increased phosphorylation of a specific protein of approximately M(r) = 30,000 (p30) in mouse osteoblastic MC3T3-E1 cells. This protein, p30, was identified as one of the small heat shock proteins (HSP) 28 from the electrophoretic pattern on two-dimensional gels, and its peptide map compared with that of heat shock-inducible p28. The increase in phosphorylation of HSP 28 seemed to correlate with growth inhibition in this cell line, since it was increased by growth inhibitory agents, such as TGF beta 1, H2O2 and 12-O-tetradecanoylphorbol-13-acetate (TPA), but not by the growth stimulating agent, epidermal growth factor (EGF), and this phosphorylation was observed only when the cells were sensitive to growth inhibition by these agents, in the late G1 phase of the cell cycle. Furthermore, in ras-transformants, whose DNA synthesis was not inhibited by these agents, this phosphorylation was not increased by these stimuli. These results indicate that phosphorylation of HSP 28 may be coupled to inhibition of DNA synthesis in this cell line.

Isolation of a gene encoding a putative leucine zipper structure that is induced by transforming growth factor beta 1 and other growth factors.

A gene sequence (TSC-22) that is induced by transforming growth factor (TGF) beta 1 was isolated by differential screening of a lambda gt10 cDNA library constructed from poly(A)+ RNA of mouse osteoblastic cells treated with TGF-beta 1 for 2 h. TSC-22 gene expression was transcriptionally activated by TGF-beta 1. It was also induced by phorbol 12-myristate 13-acetate, serum, cholera toxin, or dexamethasone, but not appreciably by epidermal growth factor. Its induction was rapid and transient, reaching a peak 2 h after TGF-beta 1 treatment, and was resistant to cycloheximide like that of c-jun. The nucleotide sequences of TSC-22 cDNA showed no homology with any known gene sequence. The open reading frame and in vitro translation product indicated that the gene encodes a polypeptide of 143 amino acids with a molecular mass of 18 kDa that contains a putative leucine zipper structure. Polyclonal antibody was raised against TSC-22 protein expressed in Escherichia coli cells, and the antibody detected a 18-kDa protein in both the cytoplasmic and nuclear fractions of [35S]Met-labeled cells. These results indicate that the TSC-22 gene is a new member of the family of early response genes, and encodes a small polypeptide that is a putative transcriptional regulator.

Inhibition of proto-oncogene c-fos transcription by inhibitors of protein kinase C and ion transport.

Both 4 beta-phorbol 12,13-didecanoate (PDD) and calcium ionophore A23187 induced c-fos mRNA accumulation with similar kinetics in human monocyte-like cells (U937). Their effects were additive. Cells pretreated with PDD were not induced to accumulate c-fos mRNA by PDD but remained responsive to the induction by A23187. Similarly cells pretreated with A23187 responded to PDD but not to A23187. Nuclear run-off transcription assay indicated that increase in the c-fos mRNA level after the second inducer treatment corresponded to transcriptional activation of the c-fos gene. The accumulation of c-fos mRNA and the transcriptional activation of c-fos induced by PDD or A23187 were inhibited by the protein kinase inhibitor H-7 and by quinidine and amiloride. The induction by A23187, but not that by PDD, was also inhibited by 4-aminopyridine, or tetraethylammonium. From these results, it is proposed that activation of protein kinase C and Na+ or K+ transport are required for c-fos induction caused by PDD and A23187.

Effects of the protein kinase C inhibitor H-7 and calmodulin antagonist W-7 on superoxide production in growing and resting human histiocytic leukemia cells (U937).

Serum, phorbol 12,13-didecanoate (PDD) and 1-oleoyl-2-acetoy-sn-glycerol (OAG) stimulated O2- release in human histiocytic leukemia U937 cells. The kinetics of O2- release caused by PDD but not by serum or OAG in growing cells differed from those in resting cells. Both the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl) 2-methylpiperidine (H-7) and calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) reduced the superoxide generation induced by these stimuli. H-7 inhibited the O2- release either from growing or resting cells but the effect of W-7 varied according to the growth phase. From these results, it is suggested that activation of protein kinase C and calmodulin-dependent process has an important role in O2(-)-release induced by serum, OAG and PDD, and that the mechanism for PDD-induced O2(-)-release is different in growing and resting cells.