Negative regulation of Pim-1 protein kinase levels by the B56beta subunit of PP2A.

The Pim protein kinases are serine threonine protein kinases that regulate important cellular signaling pathway molecules, and enhance the ability of c-Myc to induce lymphomas. We demonstrate that a cascade of events controls the cellular levels of Pim. We find that overexpression of the protein phosphatase (PP) 2A catalytic subunit decreases the activity and protein levels of Pim-1. This effect is reversed by the application of okadaic acid, an inhibitor of PP2A, and is blocked by SV40 small T antigen that is known to disrupt B subunit binding to PP2A A and C subunits. Pim-1 can coimmunoprecipitate with the PP2A regulatory B subunit, B56beta, but not B56alpha, gamma, delta, epsilon or B55alpha. Using short hairpin RNA targeted at B56beta, we demonstrate that decreasing the level of B56beta increases the half-life of Pim-1 from 0.7 to 2.8 h, and decreases the ubiquitinylation level of Pim-1. We also find that Pin1, a prolyl-isomerase, is capable of binding Pim-1 and leads to a decrease in the protein level of Pim-1. On the basis of these observations, we hypothesize that phosphorylated Pim-1 binds Pin1 allowing the interaction of PP2A through B56beta. Dephosphorylation of Pim-1 then allows for ubiquitinylation and protein degradation of Pim-1.

Hypoxia-inducible factor-2alpha regulates the expression of TRAIL receptor DR5 in renal cancer cells.

To understand the role of hypoxia-inducible factor (HIF)-2alpha in regulating sensitivity of renal cancer cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis, we transfected wild-type and mutant von Hippel Lindau (VHL) proteins into TRAIL-sensitive, VHL-negative A498 cells. We find that wild-type VHL, but not the VHL mutants S65W and C162F that do not degrade HIF proteins, cause TRAIL resistance. Knock down of the HIF-2alpha protein by RNA interference (short hairpin RNA) blocked TRAIL-induced apoptosis, decreased the level of TRAIL receptor (DR5) protein and inhibited the transcription of DR5 messenger RNA. By using luciferase constructs containing the upstream region of the DR5 promoter, we demonstrate that HIF-2alpha stimulates the transcription of the DR5 gene by activating the upstream region between -448 and -1188. Because HIF-2alpha is thought to exert its effect on gene transcription by interacting with the Max protein partner of Myc in the Myc/Max dimer, small interfering RNAs to Myc were used to lower the levels of this protein. In multiple renal cancer cell lines decreasing the levels of Myc blocked the ability of HIF-2alpha to stimulate DR5 transcription. PS-341 (VELCADE, bortezomib), a proteasome inhibitor used to treat human cancer, increases the levels of both HIF-2alpha and c-Myc and elevates the level of DR5 in renal cancer, sensitizing renal cancer cells to TRAIL therapy. Similarly, increasing HIF-2alpha in prostate and lung cancer cell lines increased the levels of DR5. Thus, in renal cancer cell lines expressing HIF-2alpha, this protein plays a role in regulating the levels of the TRAIL receptor DR5.

Induction of the proto-oncogene c-jun by angiotensin II.

Angiotensin (Ang) II causes hypertrophy of rat aortic smooth muscle cells in culture and results in the rapid activation of c-fos. This study demonstrated that Ang II also activated c-jun and, in addition, could activate the AP-1 enhancer element. These data add support for a role of Ang II as an important mediator of vascular smooth muscle cell growth.

A novel mitogen-activated protein kinase phosphatase is an important negative regulator of lipopolysaccharide-mediated c-Jun N-terminal kinase activation in mouse macrophage cell lines.

We have isolated a cDNA homologous to known dual-specificity phosphatases from a mouse macrophage cDNA library and termed it MKP-M (for mitogen-activated protein kinase phosphatase isolated from macrophages). Three other presumed splice variant isoforms have also been identified for MKP-M. The longest and most abundant mRNA contains an open reading frame corresponding to 677 amino acids and produces an 80-kDa protein. The deduced amino acid sequence of MKP-M is most similar to those of hVH-5 (or mouse M3/6) and VHP1, a Caenorhabditis elegans tyrosine phosphatase. It includes an N-terminal rhodanase homology domain, the extended active-site sequence motif (V/L)X(V/I)HCXAG(I/V)SRSXT(I/V)XXAY(L/I)M (where X is any amino acid), and a C-terminal PEST sequence. Northern blot analysis revealed a dominant MKP-M mRNA species of approximately 5.5 kb detected ubiquitously among all tissues examined. MKP-M was constitutively expressed in mouse macrophage cell lines, and its expression levels were rapidly increased by lipopolysaccharide (LPS) stimulation but not by tumor necrosis factor alpha (TNF-alpha), gamma interferon, interleukin-2 (IL-2), or IL-15 stimulation. Immunocytochemical analysis showed MKP-M to be present within cytosol. When expressed in COS7 cells, MKP-M blocks activation of mitogen-activated protein kinases with the selectivity c-Jun N-terminal kinase (JNK) >> p38 = extracellular signal-regulated kinase. Furthermore, expression of a catalytically inactive form of MKP-M in a mouse macrophage cell line increased the intensity and duration of JNK activation and TNF-alpha secretion after LPS stimulation, suggesting that MKP-M is at least partially responsible for the desensitization of LPS-mediated JNK activation and cytokine secretion in macrophages.

Effect of phorbol esters on the activity of purified transforming growth factors.

Application of either phorbol esters or transforming growth factors to normal cells in culture may induce the appearance of a malignant phenotype. Since the mechanism of transformation by both chemical tumor promoters and growth factors is unknown, it was of interest to investigate whether phorbol esters might potentiate the activity of transforming growth factors. The combination of beta-transforming growth factor (beta-TGF) and epidermal growth factor (EGF) causes the normally anchorage-dependent cells (NRK-49F) to form colonies in soft agar. In the presence of maximally stimulating concentrations of EGF and suboptimally stimulating concentrations of beta-TGF, phorbol 12-myristate-13-acetate (PMA) enhances the soft agar colony growth of NRK-49F cells in a concentration-dependent manner. However, PMA alone or in combination with either EGF or beta-TGF does not stimulate soft agar growth. In contrast to the stimulation of PMA, 4 alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, does not potentiate the effects of EGF plus beta-TGF on soft agar growth. PMA does not stimulate the growth of NRK-49F cells in monolayer, nor does it further potentiate the monolayer growth induced by EGF with or without beta-TGF. Because the addition to NRK-49F cells of compounds which potentiate beta-TGF activity increases EGF receptor number, the effects of PMA on the EGF receptor were studied. A short exposure to PMA (30 min) induces a 50% decrease in EGF receptors of NRK cells whether or not they have been exposed to beta-TGF. Scatchard analysis shows that this decrease involves primarily high affinity EGF receptors. However, cells treated with PMA for longer periods of time (4, 6, and 24 h) show no change in EGF or beta-TGF receptor binding. PMA therefore must potentiate the activity of purified TGFs without causing an increase in EGF or beta-TGF receptor binding.

Inhibition of caspases by cytokine response modifier A blocks androgen ablation-mediated prostate cancer cell death in vivo.

Androgen withdrawal is a major therapeutic modality in the treatment of prostate cancer. Although tumors initially respond, they subsequently relapse, and these recurring tumors are androgen independent. To examine possible mechanisms to explain the androgen independence of prostate cancer, we have expressed cytokine response modifier A (CrmA), a competitive inhibitor of caspases, interleukin 1beta-converting enzyme-like proteases, which mediate apoptotic cell death, in the human androgen-dependent prostate cancer cell line LNCaP. LNCaP cells require androgens for continuous growth in culture and to form tumors in nude mice. The expression of CrmA in LNCaP cells prevented the decreased growth rate induced by androgen withdrawal in tissue culture. When CrmA-expressing LNCaP (LNCaP-CrmA) cells were implanted s.c. in nude mice, the tumors grew six times faster than parental cells. Androgen ablation by castration before tumor implantation suppressed the ability of control LNCaP cells expressing nonfunctional CrmA mutant (R291T) to form tumors, but LNCaP-CrmA cells formed tumors similar in size to those formed in normal mice. When orchiectomy was performed 10 days after tumor implantation, control LNCaP cells expressing a nonfunctional CrmA mutant (R291T) regressed, but LNCaP-CrmA tumors continued to grow. Thus, inhibition of caspases prevents androgen withdrawal-induced prostate cancer cell death, suggesting that caspase activation is normally an important part of this process.

Inhibition of leukemic cell growth by the protein kinase C activator bryostatin 1 correlates with the dephosphorylation of cyclin-dependent kinase 2.

Bryostatin 1 is a natural antineoplastic agent that activates protein kinase C. Treatment of U937 human leukemic cells with bryostatin 1 caused a 60% reduction in cell growth, whereas another protein kinase C activator, phorbol myristate acetate (PMA), completely inhibited U937 cell growth. Both bryostatin 1 and PMA induced inhibition of cyclin-dependent kinase 2 (cdk2) activity. The first phase of cdk2 inhibition correlated with the transient induction of p21, a known inhibitor of cdk2. In contrast, the second phase of cdk2 inhibition correlated with the dephosphorylation of cdk2 on threonine-160, which must be phosphorylated for cdk2 activity. The level of growth inhibition induced by these two compounds correlated with the degree of cdk2 dephosphorylation as follows: bryostatin 1, 60%; PMA, 100%.

Varied differentiation responses of human leukemias to bryostatin 1.

Bryostatin 1, a macrocyclic lactone isolated from a marine bryozoan, has significant antineoplastic activity against the murine cell line P388. Like phorbol esters, bryostatin 1 is capable of binding to and activating protein kinase C, but these two compounds differ in the ability of bryostatin 1 to act as a tumor promoter. We have investigated whether bryostatin 1 can modulate the differentiated phenotype of fresh samples of human myeloid leukemia. We find that six of seven samples responded to bryostatin treatment with changes associated with a more differentiated phenotype including increases in macrophage-like morphology and an increase in adherence and OKM1 and alpha-naphthyl acetate esterase activity positivity. The percentage of cells within each sample evidencing these changes varied markedly among the seven patients' cells examined. Because of the effects of bryostatin on fresh samples we examined the ability of bryostatin to differentiate four HL-60 cell sublines obtained from different laboratories. We found that two of the cell lines did not respond either with an inhibition of growth or morphological change, while one was inhibited, and one showed both growth inhibition and some induction of macrophage-like morphology when treated with bryostatin. To test whether other differentiating agents would enhance the effects of bryostatin 1, we added tumor necrosis factor alpha and bryostatin to these four cell lines. The addition of both agents effected an additive inhibition of growth. These data suggest that bryostatin 1 alone or in combination with other biological response modifiers may have a role in the treatment of human leukemia.

In vivo administration of bryostatin 1, a protein kinase C activator, decreases murine resistance to Salmonella typhimurium.

Bryostatin 1, a potent activator of protein kinase C, has antitumor activity against murine lymphoma, leukemia, and melanoma. In vitro, this compound stimulates the release of gamma-interferon, interleukins, and hematopoietic growth factors from accessory cells and activates both T- and B-cells. Bryostatin 1 is also able to stimulate neutrophils to undergo oxidative burst and degranulation. Because of the ability of this compound to stimulate the immune system, cause release of immune mediators, and activate neutrophils, we have examined its effect on bacterial infection by using the gram-negative bacterium Salmonella typhimurium in mice. We find that animals given injections i.v. of S. typhimurium have a shortened life span if they are also given injections i.p. of nonlethal doses of bryostatin 1. There is a dose-response relationship with 100 micrograms/kg bryostatin 1 having a greater effect on survival than 40 micrograms/kg. Below 40 micrograms/kg there are no effects on survival. Analysis of the first 4 h of Salmonella infection demonstrates that bryostatin 1 does not affect the blood clearance of the bacterium. However, by day 2 of infection greater numbers of bacteria are found in the livers and spleens of mice given injections of bryostatin 1. By day 5, 10-fold more S. typhimurium bacteria are found in the livers and spleens of mice receiving 40 micrograms/kg of bryostatin 1. To determine whether bryostatin 1 was affecting growth or causing the death of bacteria, we used a Salmonella carrying a plasmid which has a temperature-sensitive origin of replication and is unable to replicate when the bacteria are in mice. This experiment demonstrates that bryostatin 1 represses bacterial killing but does not affect bacterial growth. Bryostatin 1 given i.p. stimulates a transient syndrome of weight loss and diarrhea from which the mice recover and regain weight, suggesting that bryostatin 1 may release a number of important humoral mediators in vivo. The weight loss is exacerbated by Salmonella infection with mice receiving bryostatin 1 and S. typhimurium, in that they lose approximately 33% of body weight prior to death. Thus, at doses used to treat murine tumors, bryostatin 1 treatment does not affect the clearance of S. typhimurium from the blood but does decrease the killing of bacteria in the liver and spleen, leading to early animal death. Such potential effects of bryostatin 1 on the outcome of bacterial infections should be evaluated in ongoing human trials of this agent.

In vivo administration of the anticancer agent bryostatin 1 activates platelets and neutrophils and modulates protein kinase C activity.

Bryostatin 1 is a naturally occurring macrocyclic lactone which when applied to cells in culture activates protein kinase C (PKC). In vivo bryostatin 1 functions as an anticancer agent with activity against murine lymphomas, leukemias, and melanoma. Because all organs and tissues contain PKC, normal cells would also be a likely target for this agent. Here we demonstrate that in vivo administration of bryostatin 1 activates platelets over a dose range of 0.4 to 40 micrograms/kg with half-maximal activation occurring at 3 micrograms/kg and stimulation of neutrophils over a similar dose range. This in vivo activation of neutrophils is associated with a rapid decrease in measurable cytosolic PKC, a finding consistent with translocation of the enzyme to the membrane. In contrast, no statistically significant change in PKC location was found in liver, spleen, brain, or L10A B-cell lymphoma. However, in culture the L10A lymphoma did respond to bryostatin 1 with translocation of PKC. To evaluate whether the lack of effect of bryostatin 1 on PKC in organs was secondary to rapid degradation, we developed a bioassay to measure the levels of bryostatin 1 in the blood. To measure the presence of bryostatin 1, human neutrophils were incubated with plasma from mice given injections of different concentrations of bryostatin 1. Using this assay, bryostatin 1 at levels as low as 60 nM could be measured in the plasma. A time course with this bioassay demonstrated that less than 10% of the bryostatin 1 injected was detectable after 2.5 min. These results demonstrate that bryostatin 1 is capable of activating platelets and neutrophils and modulating PKC in vivo. The lack of effect of bryostatin 1 on specific organs may be secondary to the rapid clearance/degradation of this compound from the blood.

Preclinical pharmacology of the natural product anticancer agent bryostatin 1, an activator of protein kinase C.

Bryostatin 1, a natural product anticancer agent isolated from a marine bryozoan, has been shown in tissue culture to activate protein kinase C. This agent has recently undergone Phase I testing in humans given either as a bolus i.v. injection or a continuous infusion. To understand how bryostatin 1 might be used best as an anticancer agent, a study of the pharmacokinetics, tissue distribution, metabolism, and elimination of bryostatin 1 in mice was undertaken, using [C26-3H]-labeled bryostatin 1. Following i.v. administration, the plasma disappearance curve for bryostatin 1 could be described by a two-compartment model, with half-lives of 1.05 and 22.97 h, respectively. In contrast, the plasma disappearance curve for bryostatin 1 administered i.p. was better described by a first order absorption one-compartment model, with an absorption half-life of 0.81 h and an elimination half-life of 28.76 h, respectively. The majority of radioactivity in plasma was associated with the intact drug for up to 24 h after dosing. In the first 12 h after i.v administration, urinary excretion represented the major pathway of elimination, with 23.0 +/- 1.9% (mean +/- SD) of the administered dose excreted. Within 72 h after i.v. administration, approximately equal amounts of radioactivity (40%) were excreted in feces compared to urine. Bryostatin 1 was widely distributed in many organs but concentrated in the lung, liver, gastrointestinal tract, and fatty tissue. The concentration in the gastrointestinal tract, along with the fecal excretion, suggests the possibility of enterohepatic circulation of this drug. In summary, this study demonstrates that bryostatin 1 is relatively stable in vivo, widely distributed but concentrated in some major tissues, and rapidly excreted first through urine and at later times through the feces. The data from this animal study should be useful in the design of future human trials with this anticancer drug.

Regulation of myeloid cell growth by distinct effectors of Ras.

To examine the biochemical pathways by which activated Ha-Ras(G12V) (Ha-RasV12) induces factor-independent growth of myeloid cells, Ha-Ras effector loop mutations, including Y40C, T35S, and E37G, were analysed in a mouse factor-dependent myeloid cell line, WT19. Expression of a single effector loop mutant, Ha-Ras(G12V, Y40C) (Ha-RasV12C40), inhibited factor-withdrawal apoptosis, suggesting that activation of the phosphatidylinositol 3'-kinase (Pl3K) pathway is essential to prevent cell death. Neither Ha-Ras (G12V, T35S) (Ha-RasV12S35), which activates the Rafl signaling pathway, nor Ha-Ras(G12V, E37G) (Ha-RasV12G37), which stimulates the RalGDS pathway, did not have significant effects on factor-withdrawal apoptosis of myeloid cells. Although Ha-RasV12C40 inhibited apoptosis, it did not stimulate entry into the cell cycle. Cell lines containing the combination of Ha-RasV12G37 and Ha-RasV12C40 were capable of factor-independent cell growth, while expression of the other combinations of the Ha-Ras effector mutants were not. The combined expression of Bcl-2 and Ha-RasV12G37 was not sufficient to stimulate factor independent growth, suggesting that Ha-RasV12C40 activates additional signals, besides blocking apoptosis, which are critical for factor-independent growth of myeloid cells. In factor-starved myeloid cells, inducible expression of Ha-RasV12G37 results in decreased level of p27Kip1 protein, a cyclin-dependent kinase inhibitor (CKI). These data suggest that the factor-independent growth of myeloid cells requires the activation of at least two pathways, one inhibiting factor-withdrawal apoptosis, and another causing cell cycle progression.

Constitutively active MAP kinase kinase (MEK1) stimulates SAP kinase and c-Jun transcriptional activity in U937 human leukemic cells.

Treatment of U937 human leukemic cells with the phorbol ester PMA, activates both mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK), stimulates c-Jun phosphorylation and transcriptional activity, and induces a macrophage-like differentiation of U937 cells. The involvement of the MAPK pathway in mediating both the early phosphorylation and transcriptional activation events and the chronic differentiation of U937 cells was examined utilizing constitutively active MAPK kinase (MEK1) mutants. Transient expression of an activated MEK1 construct in U937 cells was found to stimulate MAPK and SAPK activity, as well as enhancing AP1-, SRE- and c-Jun-mediated transcriptional activity. Transient transfection of MAPK phosphatase-1 (MKP-1), a protein phosphatase which preferentially dephosphorylates and inactivates MAPK, inhibited the functional effects of both PMA and the constitutively active MEK1 mutants. To determine whether specific activation of the MEK/MAPK pathway was sufficient to induce hematopoietic differentiation, U937 cell lines were established that conditionally expressed the activated MEK1 mutant under the control of the human IIa metallothionein promoter. The induction of constitutively active MEK1 protein expression resulted in an increase in MEK1 activity, c-Jun and AP-1 transcriptional activity and an inhibition of U937 cell growth. However, this growth inhibition was not accompanied by U937 cell differentiation. These results suggest that a cross-talk mechanism exists between the MAPK and SAPK signal transduction pathways in U937 cells and that PMA-mediated SAPK activation may involve the MAPK pathway. Furthermore, selective activation of the MEK/MAPK pathway utilizing a constitutively active MEK1 mutant, while growth inhibitory, was not sufficient to induce the macrophage-like differentiation of U937 cells.

Overexpression of protein kinase C beta 1 is not sufficient to induce factor independence in the interleukin-3-dependent myeloid cell line FDC-P1.

To examine whether overexpression of protein kinase C (PKC) is sufficient to allow for factor-independent growth in hematopoietic cells, we used a recombinant retroviral expression vector system to introduce a cDNA encoding the beta 1 isoform of the PKC gene into IL-3-dependent cells FDC-P1. Cell lines were generated which contained up to 24-fold increases in PKC activity. Analysis of these cell lines demonstrated that PKC activation does not play a significant role in IL-3-mediated growth control, as evidenced by the following: (1) IL-3 addition to either overexpressor cell lines did not induce morphologic changes whereas activators of PKC stimulated cellular clumping; (2) early passage FDC-P1 cells, either carrying normal or elevated levels of PKC, were not stimulated to grow by activators of PKC; and (3) addition of phorbol esters or bryostatin 1 to these cells markedly decreased the levels of PKC without affecting the ability of these cells to grow in IL-3. Therefore, we suggest that IL-3 mediated growth occurs through pathways other than involving PKC.

Bryostatin induces changes in protein kinase C location and activity without altering c-myc gene expression in human promyelocytic leukemia cells (HL-60).

When human promyelocytic leukemia cells (HL-60) are induced by phorbol esters to differentiate to macrophages, the process is accompanied by immediate activation of protein kinase C (PK-C) in the cytoplasm and later changes in DNA and RNA synthesis. Although these events are temporarily related, it remains unclear how activation of this protein kinase leads to changes in nuclear transcription. In this study, we find that bryostatin, a macrocyclic lactone which does not induce differentiation of HL-60 cells but activates PK-C, mimics the effects of phorbol esters on protein phosphorylation and PK-C location. Treatment of HL-60 cells with bryostatin stimulates phosphorylation of the surface transferrin receptor and in the cytoplasm of five proteins having the molecular weights of 17-43 kDa over the same time course as that stimulated by phorbol esters. Similarly, prolonged treatment with bryostatin, like that with phorbol esters, causes the loss of all cellular PK-C activity. Unlike the phosphorylation studies, bryostatin treatment, over a 1-100 nM concentration range and for varying lengths of time, did not affect HL-60 c-myc RNA levels, while phorbol ester treatment rapidly decreased c-myc RNA levels. These data suggest that neither the activation of PK-C and the phosphorylation of specific substrates nor the loss of total cellular PK-C activity from HL-60 cells is sufficient to induce marked decreases in c-myc levels and differentiation of HL-60 cells.

Regulation of the p58GTA cell division control-related protein kinase during phorbol 12-myristate 13-acetate-induced terminal differentiation of U937 cells.

We describe the regulation of expression, activity and subcellular localization of a cell cycle control-related kinase, p58GTA, during the withdrawal from the cell cycle of U937 human leukemic cells induced by phorbol esters. Our studies indicate that steady-state mRNA, protein levels, transcription and subcellular localization of this kinase are affected in distinctly different manners by phorbol esters (phorbol 12-myristate 13-acetate, PMA). Steady-state mRNA levels increase dramatically within 1 h of PMA treatment, while steady-state protein levels increase only slightly. However, within 24 h of PMA treatment both steady-state p58GTA mRNA and protein levels decrease markedly. Assays of p58GTA protein kinase activity show that, even though steady-state protein levels are relatively constant, protein kinase activity increases within 30 min of PMA treatment, and then peaks at 2 h and 12 h after PMA treatment. Once again, p58GTA protein kinase activity decreases by 48 h to levels similar to unstimulated cells. These results suggest that the expression of the p58GTA protein kinase gene and, quite possibly, its post-translational modification are affected by phorbol esters in a complex manner.

Co-purification of mitogen-activated protein kinases with phorbol ester-induced c-Jun kinase activity in U937 leukaemic cells.

Phorbol esters, such as phorbol myristate acetate (PMA), cause differentiation of U937 human monomyelocytic cells along the macrophage pathway. Within 15 min of PMA treatment DNA binding of the c-jun transcription factor is increased and is accompanied by rapid changes in the phosphate content of the c-jun protein. Phorbol esters stimulate phosphorylation of serines 63 and 73 located within the A1 transactivation domain of c-Jun that have previously been shown to positively regulate activity. A protein kinase activity is detectable in extracts of phorbol ester-treated U937 cells that specifically targets these two serines. Using novel assays, the protein kinase activity has been purified over 1000-fold. The major portion of protein kinase activity co-chromatographs over three columns with pp42/44 mitogen-activated protein kinases as judged by immunological methods. The significance of these results with respect to mitogen-induced transcription of AP-1-responsive genes is discussed.

Protein kinase C-independent activation of c-jun and c-fos transcription by epidermal growth factor.

Phorbol esters, epidermal growth factor (EGF) and serum induce the transient expression of the c-jun and c-fos proto-oncogenes in quiescent fibroblasts. While phorbol esters such as phorbol 12-myristate 13-acetate (PMA) are thought to induce the transcription of these genes by activating protein kinase C (PKC), the signal transduction pathway(s) mediating the effects of EGF and serum are still unclear. We have investigated whether PKC and/or calcium play a role in mediating EGF-stimulated c-jun and c-fos RNA and protein expression in quiescent NIH 3T3 fibroblasts. PMA, EGF or serum stimulated a rapid, transient increase in c-jun and c-fos expression and cJun protein synthesis in quiescent NIH 3T3 cells. Depletion of whole cell PKC activity by pretreatment with PMA abolished any subsequent response to PMA, but had no effect on the ability of EGF or serum to induce c-jun and c-fos RNA and cJun protein expression. Nuclear run-on analysis indicated that EGF-induced gene expression was due to an increase in the rate of transcription of c-jun and c-fos in both naive and PKC-depleted cells. The role of calcium in the EGF-induced expression of c-jun and c-fos was also investigated using an NIH 3T3 cell line (HER-14) overexpressing the wild type human EGF receptor. Removal of extracellular calcium by chelation with excess EGTA or use of the non-specific calcium channel blocker lanthanide, both of which abolish the EGF-induced calcium transient in HER-14 cells, had no effect on the PMA or EGF induced c-jun or c-fos response. These findings suggest that EGF induces c-jun and c-fos transcription and cJun protein synthesis in a manner independent of an increase in intracellular calcium or activation of PKC in quiescent NIH 3T3 cells.

Human neutrophils contain distinct cytosolic and particulate tyrosine kinase activities: possible role in neutrophil activation.

Tyrosine protein kinase activities were partially purified from circulating human neutrophils. Purification steps involved sequential chromatography on DEAE-Sephacel, gel filtration and affinity chromatography on a column composed of a glutamine:tyrosine copolymer linked to AH-Sepharose. The results indicate that human neutrophils contain a tyrosine kinase activity in the 150,000 x g cytosolic fraction which is distinct from the activity in a detergent extractable 150,000 x g particulate fraction. These enzyme activities are dependent on the divalent cations Mn2+ and Mg2+. Kinetics for the phosphorylation of a glutamine:tyrosine copolymer substrate demonstrated an apparent Km for the cytosolic tyrosine kinase activity of 22.3 +/- 0.3 microM, and an apparent Km for the particulate extract activity of 42.7 +/- 6.0 microM. By gel filtration chromatography, the cytosolic and particulate tyrosine kinase activities have approximate molecular masses of 80-90 and 50-60 kDa, respectively. The particulate but not the cytosolic neutrophil tyrosine kinase activity was inhibited by a novel tyrosine kinase inhibitor ST638. ST638 inhibited superoxide production in intact neutrophils stimulated with the chemotactic peptide F-Met-Leu-Phe, opsonized zymosan particles, and sodium fluoride. ST638 did not, however, inhibit superoxide production in neutrophils stimulated with phorbol myristate acetate or the calcium ionophore, A23187.

Biological activity of 26-succinylbryostatin 1.

Bryostatin 1, a macrocyclic lactone, has undergone phase I trials as an anticancer agent. Because of the lipid solubility of this compound it must be delivered either in ethanol or in a PET formulation. During the trial, these vehicles caused a large number of treatment-related side effects. We have synthesized the triethanolamine salt of 26-succinylbryostatin 1 and find that this compound is approx. 100-fold more water soluble than bryostatin 1. Because of the potential for clinical use, we have evaluated the biologic activity of this compound. We find that in a concentration-dependent manner 26-succinylbryostatin 1 is capable of activating protein kinase C (PKC) in vitro and displacing [3H]PDBu from PKC. However, at all concentrations tested the activity was less than the parent compound bryostatin 1. Addition of bryostatin 1 but not 26-succinylbryostatin 1 to U937 leukemic cells in culture stimulated a drop in cytosolic PKC, secondary to translocation of PKC to the membrane. Although 26-succinylbryostatin 1 did not stimulate a drop in the cytosolic levels of PKC, addition to U937 cells activated transcription from an AP-1 enhancer construct and c-Jun protein phosphorylation in a similar fashion to bryostatin 1 and differentiation of U937 cells. Unlike bryostatin 1, 26-succinylbryostatin 1 was unable to cause aggregation of human platelets. Although injection of bryostatin-1 into mice carrying B16 melanoma inhibits tumor growth, there was no significant inhibition of melanoma growth when identical doses of 26-succinylbryostatin 1 were injected. Therefore, 26-succinylbryostatin 1 shares some but not all of the pharmacologic properities of bryostatin 1. This compound can activate protein phosphorylation without lowering cytosolic levels of PKC.