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D-501036 [2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-N-methylpyrrole] is herein identified as a novel antineoplastic agent with a broad spectrum of antitumoral activity against several human cancer cells and an IC(50) value in the nanomolar range. The IC(50) values for D-501036 in the renal proximal tubule, normal bronchial epithelial, and fibroblast cells were >10 mumol/L. D-501036 exhibited no cross-resistance with vincristine- and paclitaxel-resistant cell lines, whereas a low level of resistance toward the etoposide-resistant KB variant was observed. Cell cycle analysis established that D-501036 treatment resulted in a dose-dependent accumulation in S phase with concomitant loss of both the G(0)-G(1) and G(2)-M phase in both Hep 3B and A-498 cells. Pulsed-field gel electrophoresis showed D-501036-induced, concentration-dependent DNA breaks in both Hep 3B and A-498 cells. These breaks did not involve interference with either topoisomerase-I and topoisomerase-II function or DNA binding. Rapid reactive oxygen species production and formation of Se-DNA adducts were evident following exposure of cells to D-501036, indicating that D-501036-mediated DNA breaks were attributable to the induction of reactive oxygen species and DNA adduct formation. Moreover, D-501036-induced DNA damage activated ataxia telangiectasia-mutated nuclear protein kinase, leading to hyperphosphorylation of Chk1, Chk2, and p53, decreased expression of CDC25A, and up-regulation of p21(WAF1) in both p53-proficient and p53-deficient cells. Collectively, the results indicate that D-501036-induced cell death was associated with DNA damage-mediated induction of ataxia telangiectasia-mutated activation, and p53-dependent and -independent apoptosis pathways. Notably, D-501036 shows potent activity against the growth of xenograft tumors of human renal carcinoma A-498 cells. Thus, D-501036 is a promising anticancer compound that has strong potential for the management of human cancers.

Mitogen activated protein kinases are of interest as research tools and as therapeutic target for certain physiological disorders. In this study, we found 2-chloro-3-(N-succinimidyl)-1,4-naphthoquinone 6 to be a selective inhibitor of MEK1 with an IC(50) of 0.38 microM. An open-chain homologue, 10, showed selective cytotoxicity against renal cancer in the NCI in vitro tumor screening. Structure-activity relationship study of eight compounds showed the cyclic imido-substituted chloro-1,4-naphthoquinone as more potent and selective MEK1 inhibitors than the open chain homologues. The imido-substituted chloro-1,4-naphthoquinones were synthesized in a straightforward fashion by refluxing 2-amino-3-chloro-1,4-naphthoquinone with the appropriate acid chloride or diacyl dichloride.

The lymphocyte-specific protein-tyrosine kinase Lck plays a critical role in T cell activation. In response to T cell antigen receptor binding Lck undergoes phosphorylation on serine residues that include serines 59 and 194. Serine 59 is phosphorylated by ERK mitogen-activated protein kinase. Recently, we showed that in mitotic T cells Lck becomes hyper-phosphorylated on serine residues. In this report, using one-dimensional phosphopeptide mapping analysis, we identify serine 59 as a site of in vivo mitotic phosphorylation in Lck. The mitotic phosphorylation of serine 59 did not require either the catalytic activity or functional SH2 or SH3 domains of Lck. In addition, the presence of ZAP-70 also was dispensable for the phosphorylation of serine 59. Although previous studies demonstrated that serine 59 is a substrate for the ERK MAPK pathway, inhibitors of this pathway did not block the mitotic phosphorylation of serine 59. These results identify serine 59 as a site of mitotic phosphorylation in Lck and suggest that a pathway distinct from that induced by antigen receptor signaling is responsible for its phosphorylation. Thus, the phosphorylation of serine 59 is the result of two distinct signaling pathways, differentially activated in response to the physiological state of the T cell.

During a search for bioactive compounds from Psorothamnus junceus, four heterocyclic compounds, psorothamnone A (1), psorothamnone B (2), dalrubone (3), and emorydone (4) were isolated from the ethanol extract of the stem bark. Psorothamnones A (1) and B (2) demonstrated inhibitory activity against protein kinase C (PKC), a key enzyme involved in the signal transduction of cell proliferation and differentiation. Dalrubone (3) and emorydone (4) showed cytotoxicity against several human tumor cell lines.

A series of beta-substituted polythiophene derivatives was synthesized through palladium-catalyzed coupling reaction. Their structure-protein kinase C (PKC) inhibitory activity relationship was studied. The carboxaldehyde and hydroxymethyl derivatives of alpha-terthiophene were potent PKC inhibitors (IC50 = 10(-7) M).

A series of alpha-terthiophene derivatives were prepared and their protein kinase C inhibitory activity were evaluated. The aldehyde derivatives were most potent inhibitors (IC50 < 1 microM). alpha-Terthiophene monoaldehyde was inactive in the inhibitions of protein kinase A, mitogen activated protein kinase and protein tyrosine kinase.

The ability of basic helix-loop-helix muscle regulatory factors (MRFs), such as MyoD, to convert nonmuscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Previous studies have shown that H-Ras 12V inhibits differentiation to a skeletal muscle lineage by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and inherent transcriptional activation properties of the proteins. To investigate the intracellular signaling pathway(s) that mediates the inhibition of MRF-induced myogenesis by oncogenic Ras, we tested two transformation-defective H-Ras 12V effector domain variants for their ability to alter terminal differentiation. H-Ras 12V,35S retains the ability to activate the Raf/MEK/mitogen-activated protein (MAP) kinase cascade, whereas H-Ras 12V,40C is unable to interact directly with Raf-1 yet still influences other signaling intermediates, including Rac and Rho. Expression of each H-Ras 12V variant in C3H10T1/2 cells abrogates MyoD-induced activation of the complete myogenic program, suggesting that MAP kinase-dependent and -independent Ras signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac1 and RhoA proteins revealed no negative effects on MyoD-induced myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 revealed that elevated MAP kinase activity is not a significant contributor to the H-Ras 12V effect. These data suggest that an additional Ras pathway, distinct from the well-characterized MAP kinase and Rac/Rho pathways known to be important for the transforming function of activated Ras, is primarily responsible for the inhibition of myogenesis by H-Ras 12V.

The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.

Purpose: Studies were conducted on oryzalin (3,5-dinitro-N,N-di(n-propyl)sulfanilamide), a widely used dinitroaniline sulfonamide herbicide, which was identified from plant extracts as an inhibitor of mitogen- and growth factor-mediated intracellular free Ca2+ ([Ca2+]i) signalling in mammalian cells., Methods and Results: Oryzalin inhibited vasopressin, bradykinin and platelet-derived growth factor [Ca2+]i signalling in Swiss 3T3 fibroblasts with IC50 values of 14, 16 and 18 microM, respectively. 45Ca2+ uptake into nonmitochondrial stores of saponin-permeabilized Swiss 3T3 cells was inhibited by oryzalin with an IC50 of 34 microM. Oryzalin inhibited colony formation of HT-29 colon carcinoma cells with an IC50 of 8 microM and inhibited the growth of a number of other cancer cell lines and primary human tumors in vitro with IC50 values in the range 3 to 22 microM. A number of oryzalin analogues were studied and an association was found between the ability to inhibit [Ca2+]i signalling and inhibition of the growth of HT-29 human colon cancer cells (P = 0.001) and of CCRF-CEM human leukemia cells (P = 0.016). Oryzalin at doses up to 600 mg/kg administered orally or subcutaneously daily to mice for 3 to 10 days beginning a day after tumor inoculation inhibited the growth of murine B16 melanoma by 63% but showed no appreciable activity when administered subcutaneously or intraperitoneally to mice beginning a number of days after tumor inoculation against a variety of human tumor xenografts. The peak plasma concentration of oryzalin following repeated subcutaneous administration of oryzalin at 600 mg/kg per day to mice was 37 microM and of its major metabolite N-depropyl oryzalin was 53 microM., Conclusion: It is unlikely that the absence of significant antitumor activity of oryzalin is a result of the inability to achieve adequate plasma concentrations.

The serine/threonine protein kinase Raf-1 is activated in response to a variety of growth factors in fibroblasts and hematopoietic cells. In T cells, Raf-1 is activated in response to stimulation through the T cell antigen receptor, the interleukin-2 receptor, and by stimulation of protein kinase C. We demonstrate here that in T cells, Raf-1 is also activated during mitosis. The mitotic activation of Raf-1 was not observed in the Lck-deficient cell line, J.CaM.1. During mitosis, Raf-1 was found to interact selectively with a mitotic form of Lck that migrated with a reduced electrophoretic mobility on SDS-polyacrylamide gels. We conclude that Raf-1 is activated during mitosis in T cells and that this mitotic activation of Raf-1 is dependent on the presence of Lck.

C3H 10T1/2 fibroblasts transformed by oncogenic ras have lower levels of protein kinase C (PKC) activity and protein. It was previously suggested that elevated levels of diacylglycerol in ras-transformed fibroblasts lead to activation-induced proteolysis of cellular PKC. We found that stable expression of T24ras in C3H 10T1/2 fibroblasts resulted in a significant decrease in levels of PKC alpha and PKC epsilon mRNA. Using C3H 10T1/2 cell lines in which the levels of activated ras can be exogenously regulated (by addition of zinc to induce the expression of a metallothionein-promoted human Ha-ras oncogene), we examined the temporal dependence of oncogenic ras expression on PKC downregulation. In these cells, downregulation of PKC protein and activity was induced but was not preceded by activation of PKC. The downregulation of PKC levels correlated with the appearance of a highly transformed morphology and was seen only at high levels of ras expression. In the inducible cells, the decrease in levels of PKC alpha mRNA had the same dependence on the levels of ras expression as did protein downregulation. These experiments provide evidence that downregulation of PKC protein levels by expression of oncogenic Ha-ras in C3H 10T1/2 fibroblasts is primarily due to altered transcriptional regulation. Because the downregulation of PKC was coupled with the onset of morphological transformation, the data suggest that this downregulation is involved in or facilitates the maintenance of a ras-transformed phenotype in C3H 10T1/2 fibroblasts.

Signal transduction is believed to be altered by cellular oncogenes or tumor suppressor genes during the transformation of normal cells into malignant cells. This proposition offers an attractive target for oncogene-based anticancer drug discovery from natural sources. Protein kinases encoded or modulated by oncogenes were used to prescreen the potential antitumor activity of medicinal plants. Protein-tyrosine kinase-directed fractionation and separation of the crude extracts of Polygonum cuspidatum and Koelreuteria henryi have led to the isolation of three different classes of protein-tyrosine kinase inhibitors, anthraquinone, stilbene and flavonoid. The anthraquinone inhibitor, emodin, displayed highly selective activities against src-Her-2/neu and ras-oncogenes.

The inhibition of phosphatidylinositol-3-kinase (PtdIns-3-kinase), protein kinase C and c-Src protein tyrosine kinase by a series of halogenated naphthoquinones and quinoline quinones related to the plant-derived naphthoquinones juglone and methyljuglone, which inhibit protein kinase C, has been investigated. Some of the compounds inhibited PtdIns-3-kinase at micromolar concentrations and below. PtdIns-3-kinase inhibition was time dependent and could be prevented by endogenous thiol. The compounds were only weak inhibitors of PtdIns-4-kinase. Some of the compounds inhibited protein kinase C, but c-Src protein tyrosine kinase was only weakly inhibited. In intact cells, PtdIns-3-kinase was only partly inhibited by concentrations of the halogenated quinones that inhibited cell growth. Some halogenated quinones showed in vivo antitumor activity without accompanying toxicity, while methyljuglone was without in vivo antitumor activity. Halogenated quinones may have multiple biochemical effects in the cell that could contribute to their cytotoxic and antitumor effects. Inhibition of PtdIns-3-kinase by the halogenated quinones may provide a lead for the development of more potent and specific inhibitors.

The defects in the regulation of cell growth and differentiation that manifest themselves as cancer result from multiple defective genes and their products, which are involved in the processes of cellular signaling, regulation of gene expression and control of the cell through its replication cycle. Each of these molecular defects represents a new target for development of novel therapeutic agents and prophylactic interventions. Evidence suggests that such therapeutic agents will show great efficacy for cells made cancerous by the single targeted defect. However, poor anticancer efficacy for clinically presenting cancer may occur as a result of the multiple molecular lesions. A combined-agent approach seems likely to be more successful, but this will require diagnosis of each tumor in substantially greater detail, down to the molecular level. When such molecular diagnosis becomes generally feasible, it should be possible to use combinations of highly specific agents at very low doses for therapy and ultimately for prevention of tumor metastasis. Chemoprevention in general may be achieved more easily than therapy with mechanism-based interventions, as certain individual lesions, in theory, may be rate limiting for carcinogenesis but may not be a significant contributor to the neoplastic phenotype by the time the tumor presents in the clinic.

The transfer of membranes from the endoplasmic reticulum to the Golgi apparatus occurs via 50-70 nm transition vesicles which derive from part-rough, part-smooth transitional elements of the endoplasmic reticulum (TER). Vesicle budding from the TER is an ATP-dependent process both in vivo and in vitro. An ATPase with a monomer molecular weight of 100 kD by SDS-PAGE has been isolated from TER and designated as TER ATPase. The native TER ATPase has been characterized as a hexamer of six 100-kD subunits by gel filtration. The protein catalyzes the hydrolysis of [gamma 32-P]ATP and is phosphorylated in the presence of Mg2+. It is distinct from the classical transport ATPases based on pH optima, ion effects, and inhibitor specificity. Electron microscopy of negatively stained preparations revealed the TER ATPase to be a ring-shaped structure with six-fold rotational symmetry. A 19-amino acid sequence of TER ATPase having 84% identity with valosin-containing protein and 64% identity with a yeast cell-cycle control protein CDC48p was obtained. Anti-synthetic peptide antisera to a 15-amino acid portion of the sequence of TER ATPase recognized a 100-kD protein from TER. These antisera reduced the ATP-dependent cell-free formation of transition vesicles from isolated TER of rat liver. In a reconstituted membrane transfer system, TER ATPase antisera inhibited transfer of radiolabeled material from endoplasmic reticulum to Golgi apparatus, while preimmune sera did not. The results suggest that the TER ATPase is obligatorily involved in the ATP requirements for budding of transition vesicles from the TER. cDNA clones encoding TER ATPase were isolated by immunoscreening a rat liver cDNA library with the affinity-purified TER ATPase antibody. A computer search of deduced amino acid sequences revealed the cloned TER ATPase to be the rat equivalent of porcine valosin-containing protein, a member of a novel family of ATP binding, homo-oligomeric proteins including the N-ethylmaleimide-sensitive fusion protein.

A convenient and reliable multisample assay for the screening of inhibitors of the growth factor signalling enzyme phosphatidylinositol-3-kinase (PtdIns-3-K) has been developed. Four natural product inhibitors of Ptdlns-3-K have been identified with IC50 values for hypericin 0.18 microM, emodin 3.3 microM, asperuloside 2.0 microM and uttronin A 1.1 microM.

A convenient and reliable multisample assay for the screening of inhibitors of the growth factor signalling enzyme phosphatidylinositol specific phospholipase C (PtdInsPLC) has been developed. Three naturally occurring peptide inhibitors of PtdInsPLC have been identified, myroridin K, streptothricin B and edeine, with IC50 values of 8.3, 6.7 and 16.1 microM, respectively. All three peptides inhibited colony formation of HT-29 human colon adenocarcinoma cells, with IC50 values of 7.2, 3.9 and 13.0 microM, respectively. The compounds also inhibited the growth of other human cancer cells in culture. One of the peptides, myroridin K, has previously been reported to have in vivo antitumour activity. It is possible that inhibition of PtdInsPLC is responsible for the cell growth inhibition and antitumour properties of the peptide compounds.

The antitumor sesquiterpene lactone helenalin, which is found in species of the plant genus Helenium, caused a marked potentiation of the increases in intracellular free Ca2+ concentration ([Ca2+]i) produced by mitogens such as vasopressin, bradykinin, and platelet-derived growth factor in Swiss mouse 3T3 fibroblasts. Removing external Ca2+ partly attenuated the increased [Ca2+]i responses caused by helenalin. The increased [Ca2+]i responses occurred at concentrations of helenalin that inhibited cell proliferation. At higher concentrations, helenalin inhibited the [Ca2+]i responses. No change in resting [Ca2+]i was caused by helenalin even at high concentrations. Other helenalin analogues also increased the [Ca2+]i response. Helenalin did not inhibit protein kinase C (PKC) and PKC appeared to play a minor role in the effects of helenalin on [Ca2+]i responses in intact cells. Studies with saponin-permeabilized HT-29 human colon carcinosarcoma cells indicated that helenalin caused an increased accumulation of Ca2+ into nonmitochondrial stores and that the potentiating effect of helenalin on mitogen-stimulated [Ca2+]i responses was due in part to an increase in the inositol-(1,4,5)-trisphosphate-mediated release of Ca2+ from these stores.

Bioassay-directed fractionation of a medicinal plant, Polygonum cuspidatum (Polygonaceae), has led to the discovery of a hydroxystilbene, resveratrol [1], as an inhibitor of a protein-tyrosine kinase (p56lck) partially purified from bovine thymus. Both trans and cis isomers of resveratrol possess comparable protein-tyrosine kinase inhibitory activity. Comparison of the IC50 values of resveratrol for protein-tyrosine kinase inhibitory activity with those of piceid (resveratrol-O3-beta-glucoside) [2] and resveratrol-O4'-beta-glucoside [3] shows the requirement of free hydroxyl groups on both phenyl rings for the protein-tyrosine kinase inhibition. Protein kinase C inhibitory analysis suggests the requirements of two free hydroxyl groups on one phenyl ring only.

N-Myristoylation of p56lck, a member of the Src family of protein-tyrosine kinases, is essential for its proper targeting to the plasma membrane. 2-Hydroxymyristic acid (HMA) is an analog of myristic acid that becomes metabolically activated in cells to form 2-hydroxymyristoyl-CoA, a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that catalyzes protein N-myristoylation [Paige, L. A., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (1990) Biochemistry 29, 10566]. In the presence of HMA, LSTRA cells, which overexpress p56lck, synthesized nonmyristoylated p56lck, which displayed a reduced electrophoretic mobility on SDS-polyacrylamide gels identical to that of a nonmyristoylated Gly2-->Ala2 mutant of p56lck. Treatment with myristic acid, 2-hydroxypalmitic acid, or 2-fluoromyristic acid did not result in the synthesis of nonmyristoylated p56lck. In contrast to the membrane-associated, myristoylated p56lck, nonmyristoylated p56lck was cytosolic. Although nonmyristoylated p56lck retained tyrosine kinase activity, it was not labeled in vivo with [32P]orthophosphate, indicating that a change in subcellular location altered its state of phosphorylation. A pulse-chase analysis revealed that cytosolic, nonmyristoylated p56lck was less stable than the myristoylated enzyme. In cell lines that do not overexpress p56lck, HMA treatment resulted in a reduction in the levels of both newly synthesized and total p56lck. Treatment of CD4+ cells with HMA caused a corresponding decrease in the amount of CD4-associated p56lck. Thus, chemical inhibition of protein N-myristoylation with HMA is an effective method for reducing the amount of p56lck available at the plasma membrane for signal transduction.

Phosphorylation of protein kinase C (PKC) may be an important mode of regulation of this enzyme that plays a key role in mouse skin tumor promotion and in mammalian cell signal transduction. To investigate this possibility, PKC was specifically immunoprecipitated from Abelson murine leukemia virus-transformed normal rat kidney cells that had been metabolically labeled with [32P]orthophosphoric acid. The Mr 80,000 phosphoprotein that was specifically immunoprecipitated from Abelson murine leukemia virus-transformed normal rat kidney cells was found to be identical with purified rat brain PKC that had undergone cell-free autophosphorylation. This is based on comparisons of peptides generated by partial proteolysis with Staphylococcus aureus V8 protease by one-dimensional polyacrylamide-sodium dodecyl sulfate gel electrophoresis and of tryptic peptides by reversed-phase high-pressure liquid chromatography. These data are consistent with phosphorylation of PKC in cells having occurred via autophosphorylation. The autophosphorylation of PKC was stimulated by treatment of C3H 10T1/2 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate or sn-1,2-dioctanoylglycerol. Exposure of cells to 100 nM 12-O-tetradecanoylphorbol-13-acetate for 15 min increased the phosphorylation of PKC by 5-fold in the particulate fraction, while treatment with 100 microM dioctanoylglycerol enhanced phosphorylation of PKC only by 2-fold. Phosphorylation of PKC in response to activation may have significance for altering the sensitivity of PKC to proteolytic down-regulation and/or to subsequent activation.

Expression of the oncogenic form of H-ras p21 in the mouse myogenic cell line, 23A2, blocks myogenesis and inhibits expression of the myogenic regulatory factor gene, MyoD1. Previous studies from a number of laboratories have demonstrated that the activation of ras p21 is associated with changes in phospholipid metabolism that directly, or indirectly, lead to elevated levels of intracellular diacylglycerol and the subsequent activation of protein kinase C (PKC). To assess the importance of PKC activity to the ras-induced inhibition of skeletal myogenesis, we examined the levels of PKC activity associated with the terminal differentiation of wild-type myoblasts and with the differentiation-defective phenotype of 23A2 ras cells. We demonstrate that there is a 50% reduction in PKC activity during normal myogenesis and that PKC activity is required for myoblast fusion, but not for the transcriptional activation of muscle-specific genes. In contrast, we found that the differentiation-defective 23A2 ras cells possess two- to threefold more PKC activity than wild-type myofibers and that reducing the PKC activity in these cultures does not reverse their non-myogenic phenotype. On the other hand, if PKC activity is downregulated in 23A2 cells before the expression of activated ras p21, myogenesis is not inhibited. These results suggest that activated ras p21 relies on a PKC-dependent signal transduction pathway to initiate, but not to sustain, its negative effects on 23A2 skeletal myogenesis and underscore the potential importance of PKC activity to the proper control of skeletal muscle differentiation.

The present study has characterized several aspects of the mouse epidermal protein kinase C (PKC) system and compared phorbol ester-sensitive and -resistant mice. Protein immunoblots of partially purified epidermal PKC preparations from SENCAR and C57BL/6 mice indicated the presence of the gamma-, beta-, and alpha-isozymes of PKC in both strains. Hydroxylapatite chromatography profiles of epidermal PKC isozymes from SENCAR and C57BL/6 mice revealed three major peaks of PKC activity eluting in fractions similar to those observed in chromatograms of brain tissue and corresponding to PKC-gamma, -beta, and -alpha. Further analyses of hydroxylapatite chromatography fractions revealed that PKC-gamma and -beta were present in approximately similar proportions and were much more abundant than PKC-alpha. This distribution of epidermal PKC isozymes was similar in both strains. After a single topical application of 3.4 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) to SENCAR mouse epidermis, total PKC activity in the cytosol fraction decreased rapidly to about 50% of control within 15 min and was accompanied by an increase (approximately 150% of control) of PKC activity in the membrane fraction. At 4 h, PKC activities were significantly lower than the control levels and remained downregulated through 96 h with a maximal decrease (to approximately 25-30% of the control) in both cytosol and membrane fractions at h. PKC activity returned to control levels by 168 h. Ca++/phospholipid-independent kinase activity was the same as control levels at 15 min, 1 h, and 4 h after TPA treatment but was elevated above control levels at 24 h, 48 h, and 96 h, and by 168 h returned essentially to control levels. No differences were found in the magnitude or kinetics of TPA-induced translocation and downregulation of total PKC or appearance of Ca++/phospholipid-independent kinase activity between SENCAR, DBA/2, and C57BL/6 mice. Scatchard analyses using a two binding site model revealed that the apparent Kd and Bmax values for binding of PDBu to epidermal cytosol and membrane fractions were similar between SSln, SENCAR, DBA/2, and C57BL/6 mice. The present results demonstrate for the first time that mouse epidermis contains significant amounts of the three major PKC isozymes that are present in brain, especially PKC-gamma. In addition, topical application of a promoting dose of TPA did not lead to complete loss of PKC activity in either the membrane or cytosol fractions of mouse epidermis. In conclusion, no differences were observed between phorbol ester-sensitive and -resistant mice in any aspect of epidermal PKC examined.

Mouse skin has a long history as a useful model for the study of the mechanism of carcinogenesis (6). In particular, the availability of specific diterpene esters has made possible rapid progress in understanding the mechanism of tumor formation (4,6,8,19,36,41), although certain details may be unique to promotion by phorbol esters. Evidence is compatible with an essential role for elevated levels of polyamines in tumor promotion, but other components of phorbol ester action on mouse skin are also essential (27,40,54). These may include the production of dark cells (22), inhibition of maturation (2,19,41), and the elimination of metabolic cooperation (12,57). Factors modifying biochemical processes that are essential to tumor formation produce a parallel effect on tumor formation. Some of these inhibitors act synergistically to inhibit tumor formation (50,55), and knowledge of their action may lead to practical application for the prevention of human cancer.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) binds reversibly and with high affinity and specificity to nuclear macromolecules in mouse epidermis. The dissociation constants determined from Scatchard analysis of epidermal nuclei and nuclear macromolecules are 3.58 +/- 0.66 (S.E.) and 2.18 +/- 0.54 nM, respectively. The solubilization of TPA receptors from epidermal nuclei by DNase I was examined. Following a 20-min digestion at 22 degrees, more than a 2-fold increase in specific TPA binding was observed in the supernatant relative to non-nuclease-treated nuclei (0.71 versus 0.32 pmol/mg protein, respectively). Our data indicate that epidermal nuclei contain saturable and specific TPA-binding macromolecules and that these binding components may be associated with regions of chromatin that are preferentially susceptible to nucleolytic cleavage. These data suggest the existence of nuclear receptors for the phorbol ester tumor promoters. These observations may necessitate a more critical assessment of plasma membrane binding as the sole binding site responsible for triggering the multistep process of tumor promotion in mouse epidermis.

Bryostatin 1 (Bryo), a macrocyclic lactone, stimulates some but not all of the biologic effects which are induced by phorbol esters (PEs). In vitro, it competes with PEs for binding to whole cells and activates the calcium/phospholipid-dependent protein kinase, PK-C. To examine whether Bryo, like PEs, is able to stimulate the nonadherent growth of cells, we used the mouse epidermal cell line JB6, which is stimulated by PEs to grow in soft agar. Like PEs, Bryo stimulates both the adherent and nonadherent growth of these cells, but Bryo (0.001-1 microM) is less active than equivalent concentrations of PEs. To attempt to explain the biologic differences between these two agents, we examined the modulation of PK-C by both PEs and Bryo. In a phosphotransferase assay using partially purified PK-C from JB6 cells, Bryo (1-0.001 microM) stimulated less phosphorylation of histone substrate than did PMA. Also, when whole cells were treated with equal concentrations of Bryo or PMA, Bryo stimulated a decreased loss of PK-C from the cytosol. Using purified isozymes of PK-C from rat brain, Bryo demonstrated identical competition to PMA for binding to forms alpha and gamma but decreased binding to form beta. Hydroxylapatite chromatography of JB6 cytosol demonstrated that these cells contain largely peak 2, or beta-PK-C. Although Bryo more weakly activates PK-C from JB6 cells, prolonged exposure of JB6 cells to either 1.0 or 0.01 microM Bryo caused a more rapid loss of immunologically detectable PK-C than did similar concentrations of PEs. We conclude that Bryo is capable of stimulating both the nonadherent and the adherent growth of JB6 cells in a similar fashion to phorbol esters. The differences in biologic effects of Bryo and PMA may be partially explained by Bryo's modulation of PK-C.

Tumor-promoting phorbol esters activated a calcium-activated, phospholipid-dependent protein kinase by direct complexation with this protein kinase and phospholipid in the presence of divalent cations and this complexation was identical to association of kinase/receptor activity with cell membranes. Treatment of isolated mouse spleen lymphocytes with phorbol ester tumor promoters resulted in a rapid shift in the subcellular localization of both the phorbol ester receptor and a calcium-activated, phospholipid-dependent protein kinase activity. Both activities shifted from almost entirely soluble to largely membrane-associated, which is consistent with a single protein possessing both activities. Activation of partially purified kinase/receptor activity by phorbol ester or calcium alone or in combination occurred in parallel to the formation of a complex between the kinase/receptor and phospholipid. Magnesium also was important both for complex formation and for activation of the protein kinase. Although phorbol ester did not appear to affect the affinity of the kinase/receptor for phospholipid, it did increase the extent of formation of a stable complex between the receptor and the phospholipid. These observations support the hypothesis that the cell membrane is the locus of action of both the phorbol esters and the calcium-activated, phospholipid-dependent protein kinase activity.

Biochemical and immunological comparison of mouse C3H 10T 1/2 fibroblasts and C3H 10T 1/2 fibroblasts transfected with human activated Ha-ras oncogene indicated significantly lower levels of protein kinase C (PKC) activity and protein in the ras-transfected cells. This effect was observed in three clonal cell lines transfected with an activated ras oncogene. Cytosolic extracts of the ras-transfected cells contained calcium-activated, phospholipid-dependent protein kinase (PKC) activity at 61% of the level of activity present in C3H 10T 1/2 cells. A similarly decreased level of phorbol ester-binding activity was observed in these cells. Analysis of the subcellular distribution of PKC activity in cells failed to indicate significant differences between these cell lines. Immunoblots showed a lower abundance of the Mr 80,000 PKC in ras-transfected cell homogenates and extracts compared to C3H 10T 1/2 cells. Both C3H 10T 1/2 cells and cells transfected with ras expressed only one of the PKC isozymes as resolved by hydroxylapatite chromatography demonstrating that ras transfection of cells did not induce expression of alternative PKC isozymes. These observations indicate that PKC was partially down-regulated in ras-transfected cells, perhaps resulting from constitutively elevated levels of products of phosphatidylinositol-4,5-bisphosphate hydrolysis. Although C3H 10T 1/2 cells were previously shown to be distinct from NIH 3T3 cells in their sensitivity to transformation by the T24-ras oncogene, ras transformation appears to partially down-regulate PKC in C3H 10T 1/2 cells in a manner identical to that for ras-transformed NIH 3T3 cells. This indicates that down-regulation of PKC directly results from the expression of an activated ras oncogene independently of cellular sensitivity to transformation by expression of ras. The common action of ras transformation and phorbol esters to down-regulate PKC provides a possible mechanism for synergism during multistage carcinogenesis.

Retinoic acid, a potent inhibitor of mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate, fails to inhibit tumor formation by the complete carcinogen, 7, 12-dimethylbenz[a]anthracene (DMBA). To obtain further clues about the nature of the mechanism of the carcinogenic process as well as the mechanism of the effect of retinoic acid on tumor promotion, the effect of retinoic acid and two other modifiers (dexamethasone and 7,8-benzoflavone) of tumor formation on tumor promotion by 7-bromomethylbenz[a]anthracene (BrMBA) was determined. BrMBA, a structural analogue of DMBA, is a weak mouse skin tumor-initiating agent but is a good skin tumor promoter. Application of 10, 100, and 200 nmol of BrMBA twice weekly to DMBA-initiated skin resulted in 0, 1.6, and 2.5 papillomas per mouse, and 0, 44, and 60% of mice had papillomas at the 25th week of promotion treatment, respectively. Application of 17 nmol of retinoic acid or 76 nmol of dexamethasone 30 min prior to each twice weekly application of 100 nmol of BrMBA to DMBA-initiated skin inhibited the formation of skin papillomas by 73 and 100%, respectively. 7,8-Benzoflavone, at a 367-nmol dose, did not inhibit tumor promotion by BrMBA. Application of 200 nmol of BrMBA to mouse skin induced epidermal ornithine decarboxylase activity; a peak activity was observed between 8 and 18 hr following BrMBA treatment. Application of 17 nmol of retinoic acid or 76 nmol of dexamethasone inhibited the induction of ornithine decarboxylase activity by BrMBA. 7,8-Benzoflavone did not inhibit the induction of ornithine decarboxylase activity by BrMBA. Retinoic acid and dexamethasone, which inhibit tumor promotion by 12-O-tetradecanoylphorbol-13-acetate, also inhibited tumor promotion by BrMBA, but the nature of the mechanism of tumor promotion by BrMBA is unclear; BrMBA did not inhibit specific binding of 12-O-[3H]tetradecanoylphorbol-13-acetate to the cellular membrane fraction of mouse epidermis.

Data from three different sorts of experiments that were designed to provide additional information on the mechanism of tumor formation are presented. The aim of the first approach was to obtain further evidence for the possible relevance of the induction of ornithine decarboxylase (ODC) activity to tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin. The irreversible inhibitor of ODC activity, alpha-difluoromethylornithine, not only prevented ODC activity in a dose-dependent manner following skin application, it also prevented skin tumor promotion by TPA. Because in tumor promotion experiments TPA must be applied repetitively in order to elicit tumors, the effect of various time intervals between two or more applications on ODC induction and polyamine levels was investigated. Double applications at intervals greater than 48 hr led to a larger induction of ODC than after a single application. In contrast, at intervals of 24 hr or less, the first application caused a refractory state; the second application did not induce ODC activity even at times after ODC activity had returned to the very low, control activity. The aim of the third approach was to more directly determine the function of TPA in the promotion process by identifying the receptor(s) for TPA and to ascertain the function of the receptor(s). A receptor was purified from the particulate protein fraction of mouse brain and it was found that divalent calcium and phosphatidylserine were essential for the maintenance of binding activity during purification. Furthermore the receptor copurified with protein kinase C; it may be that a TPA receptor is protein kinase C and that TPA activates phosphorylating activity.

Protein kinase activity copurified with the receptor for 12-O-[3H]tetradecanoylphorbol-13-acetate during its purification from mouse brain particulate protein. All attempts to resolve the protein kinase activity from the receptor were unsuccessful. The isolated receptor required phospholipid and divalent calcium for maximal protein kinase activity. The protein kinase was not activated by cyclic nucleotides or calmodulin. There are striking similarities between the receptor-associated protein kinase activity and the calcium- and phospholipid-dependent protein kinase, which has been suspected of mediating the effects of biological stimuli associated with increased phosphatidylinositol turnover.

The KG-1 myeloid leukemia cell line differentiates into macrophages in response to 12-O-tetradecanoylphorbol 13-acetate (TPA), while its spontaneous variant, the KG-1a cell line, is resistant to the differentiative effects of TPA. To determine the mechanism underlying these differential responses to TPA, experiments were performed to determine the relative numbers of TPA binding sites, protein kinase C (PKC) enzyme activity levels, PKC translocation responses and PKC gene expression in these cell lines. KG-1a cells exhibited 40% fewer high affinity binding sites for TPA than KG-1 cells. Although PKC translocation from cytosol to the membrane fraction was observed in both cell types, total PKC activity, measured in vitro using type III-S histone as substrate, was reduced by 70% in KG-1a cells. These biochemical differences between the parental line and the phorbol ester non-responsive variant were correlated with the depressed level of PKC-beta RNA abundance in KG-1a cells. Both lines expressed PKC-alpha RNA at comparable levels. Chronic exposure to TPA resulted in down-regulation of PKC enzyme activity in both cell lines, and a selective decrease in PKC-beta RNA transcripts in both cell types. In contrast, chronic TPA treatment had no effect on the levels of PKC-alpha RNA in KG-1 and KG-1a cells. Our results indicate a correlation between the level of PKC-beta expression and the responsiveness of myeloid lineage precursor cells to the differentiative effects of TPA.

The protein that specifically binds 12-O-[3H]tetradecanoyl-phorbol-13-acetate was solubilized from mouse brain particulate protein with detergents and purified to apparent homogeneity by chromatograph on diethylaminoethyl-cellulose, hydroxylapatite, phenyl agarose, and gel filtration. The purified binding activity consisted of a single polypeptide of apparent molecular weight of 70,000. The isolated receptor protein required reconstitution with phospholipid and cations for binding. The solubilization, isolation, and reconstitution of the receptor did not alter its relative affinity for several different phorbol esters. The isolation of the receptor will aid the determination of both its biological function and its extent of involvement in tumor promotion and cellular physiology.

A soluble cytosolic protein which specifically and with high affinity binds tumor promoting phorbol ester tumor promoters was found in mouse tissues Calcium and phosphatidyl serine had to be included in the binding assay to obtain maximal binding activity by the soluble receptor. Soluble binding activity levels paralleled the levels of membrane associated binding activity with the soluble activity accounting for 27% to 80% of the total. Except the calcium and phospholipid requirement, no difference was found between the soluble and membrane associated receptors, suggesting that they are the same protein which can move between the soluble cytosol and cell membranes.