An inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine(H-7) inhibits TPA-induced reduction of vincristine uptake from P388 murine leukemic cell

The effects of protein kinase C inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) on tumor-promoting phorbol ester induced inhibition of vincristine uptake in P388 murine leukemic cells were investigated with the objective of assessing the possible role of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) in vincristine uptake. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a potent inhibitor at concentrations above 1 nM. Other phorbol esters also inhibited vincristine uptake in approximate proportion to their activity in competing for [20-3H]phorbol 12,13-dibutrate binding. TPA enhanced the Ca2+-activated, phospholipid-dependent phosphorylation of histone III-S by a soluble protein fraction of cells. Phosphorylation of various cell lysate proteins (p18, p21, p29, p34 and p45) were also stimulated by TPA. These TPA-induced stimulations were also inhibited dose-dependently by H-7. It is tentatively concluded that the phosphorylation of cell lysate protein substrates by protein kinase C may be an important mechanism linked to the regulation of vincristine uptake in leukemic cell.     

Inhibition of phorbol ester-induced phenotypic differentiation of HL-60 cells by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor

To identify the possible role of calcium ions in cell differentiation, we studied the extracellular Ca2+ requirement and the effect of Ca2+/phospholipid-dependent protein kinase (protein kinase C) inhibitor on proliferation and differentiation of human promyelocytic leukemic HL-60 cells. HL-60 cells grew equally well in 0.1 and 1.0 mM Ca2+ media. The addition of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), 1,25-dihydroxyvitamin D3, and all-trans-beta-retinoic acid inhibited the cell growth and induced mature macrophage and granulocyte phenotypes in 1.0 mM Ca2+ medium. 1,25-Dihydroxyvitamin D3 and all-trans-beta-retinoic acid induced HL-60 differentiation to the same degree in 0.1 mM Ca2+ and 1.0 mM Ca2+ media. However, TPA failed to induce HL-60 differentiation or to inhibit proliferation in a 0.1 mM Ca2+ medium. The decrease of extracellular Ca2+ from 1.0 to 0.1 mM caused a significant drop in the intracellular Ca2+ level in undifferentiated and TPA-treated HL-60 cells, although no rapid change in cytosolic Ca2+ was detected in response to TPA addition. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor, inhibited proliferation of HL-60 cells in a dose-dependent manner. In contrast, H-7 selectively restored the proliferation of TPA-treated HL-60 cells and inhibited TPA-induced phenotypic differentiation. However, the same concentrations of 1-(5-isoquinolinylsulfonyl)-2,3-dimethylpiperazin and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, analogues of H-7 that inhibit protein kinase C more weakly, had no effect on the proliferation or differentiation induction. H-7 also suppressed 1,25-dihydroxyvitamin D3- and all-trans-beta-retinoic acid-induced phenotypic changes of HL-60 cells but did not eliminate the growth inhibition by these inducers. These results demonstrate the Ca2+ requirement and the protein kinase C involvement in phorbol ester-induced phenotypic differentiation of HL-60 cells.     

Activation of MDR1 (P-glycoprotein) gene expression in human cells by protein kinase C agonists.

P-glycoprotein, encoded by the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump for various lipophilic compounds. MDR1 is expressed in several types of normal human tissues and in a variety of tumors, where its expression has been correlated with resistance to chemotherapy. Some P-glycoprotein-overexpressing multidrug-resistant cell lines contain elevated amounts of protein kinase C (PKC). PKC activation was shown to increase the level of drug resistance in several cell lines, but the functional association of PKC with P-glycoprotein-mediated multidrug resistance remains unclear. We have studied the effects of lymphocyte-activating agents on P-glycoprotein activity in normal human lymphocytes, and found that 12-O-tetradecanoylphorbol-13-acetate (TPA), an efficient agonist of PKC, increased the activity as well as the levels of P-glycoprotein in these cells. TPA also increased P-glycoprotein expression in several cell lines derived from different types of leukemias and solid tumors. The increase in MDR1 gene expression was observed at both the protein and RNA levels. Induction of MDR1 mRNA was apparent as early as two hours after the addition of TPA. Diacylglycerol (DAG), a physiological stimulant of PKC, also increased the expression of MDR1 mRNA and P-glycoprotein. The induction of MDR1 expression by TPA and DAG was suppressed by staurosporine, a protein kinase inhibitor. The results suggest that MDR1 gene expression in different cell types is regulated by a PKC-mediated pathway. This finding has implications for the emergence of multidrug resistance in vitro and in vivo.     

Evidence for involvement of the protein kinase C pathway in the activation of p37v-mos protein kinase

Protein kinases are known to undergo phosphorylation to regulate their activity. To determine whether the protein kinase activity of p37v-mos was similarly regulated, we investigated the influence of two well known protein kinases, namely protein kinase C and protein kinase A, on the activity of p37v-mos in vivo. NIH3T3 cells chronically transformed with Moloney murine sarcoma virus 124 were treated with high concentrations (200-400 nM) of phorbol 12-myristate 13-acetate (PMA) for 24-48 h, concentrations known to result in the total loss of protein kinase C by causing its translocation from the cytosol to cell membranes where it is downregulated. PMA treatment caused a drastic decrease in the protein kinase activity of p37v-mos without affecting its steady state level. Similar results were obtained with p85gag-mos expressed in ts110 Mo-MuSV transformed NRK cells. Control treatment with an inactive analogue of PMA, 4-alpha phorbol 12,13-didecanoate, had no effect on the p37v-mos protein kinase activity. Treatment of cells with a direct chemical inhibitor of protein kinase C, H-7 (1-(5-isoquinoline sulfonyl)-2-methylpiperazine dihydrochloride), approximately halved p37v-mos kinase activity, although the drug did not inhibit p37v-mos kinase activity directly in vitro. In contrast to the PMA effect, in vivo activation of protein kinase A by 8-(4-chlorophenylthio)-adenosine 3',5' cyclic monophosphate did not affect p37v-mos protein kinase activity levels. These findings indicate that the protein kinase C pathway but not the protein kinase A pathway modulates v-mos protein kinase activity.     

Inhibition of MCF-7 cell growth by 12-O-tetradecanoylphorbol-13-acetate and 1,2-dioctanoyl-sn-glycerol: distinct effects on protein kinase C activity

We have investigated the effects of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and permeant diacylglycerol 1,2-dioctanoyl-sn-glycerol (DiC8) on MCF-7 cell proliferation and protein kinase C activity. DiC8 mimics the effects of TPA on both cell morphology and proliferation, with an ED50 value of 11 micrograms/ml for cell growth inhibition. As with TPA and phorbol 12,13-dibutyrate, DiC8 enhances the degree of phosphorylation of an endogenous Mr 28,000 protein in a time- and dose-dependent manner. The effect is measurable upon 5 min of cell treatment with each protein kinase C activator and reaches a maximum at 30 min. The ED50s observed are 5 ng/ml and 20 micrograms/ml, respectively, for phorbol esters and DiC8. The Mr 28,000 protein is found in the cytosolic fraction and is phosphorylated on serine residues by both TPA and DiC8. Further characterization of the phosphorylated proteins using a highly resolutive two-dimensional electrophoresis demonstrates that the two-protein kinase C activators lead to slightly distinct protein phosphorylation patterns with an extra set of proteins phosphorylated under TPA but not DiC8 stimulation. Contrary to TPA, DiC8 induces only a partial and transient translocation of protein kinase C activity from the cytosolic to the particulate compartment. Moreover, no down-regulation of protein kinase C is observed after prolonged treatment of MCF-7 cells with DiC8, while only 10% of the initial protein kinase C level remains present in cells treated with TPA for 48 h. However, this remainder enzymatic activity is sufficient to induce the phosphorylation of the Mr 28,000 protein at its maximal level. In conclusion, our results reinforce the hypothesis of a negative modulatory role of protein kinase C in MCF-7 cell proliferation but suggest that the two activators TPA and DiC8 could induce distinct molecular events with regard to the enzyme recruitment and activity as well as to its further processing.     

Enhanced nuclear diacylglycerol kinase activity in response to a mitogenic stimulation of quiescent Swiss 3T3 cells with insulin-like growth factor I

Results from several laboratories have established the existence in the nucleus of an autonomous polyphosphoinositide cycle, which is involved in both cell proliferation and differentiation. A key step of intranuclear polyphosphoinositide metabolism is the phospholipase C-mediated generation of diacylglycerol (DAG). In insulin-like growth factor (IGF)-I-stimulated Swiss 3T3 cells, a transient elevation of intranuclear DAG levels is essential for attracting the alpha isoform of protein kinase C (PKC) to the nucleus. Previous evidence has shown that the nucleus also contains DAG kinase, i.e., the enzyme that yields phosphatidic acid from DAG, thus terminating PKC-mediated signaling events. Here we show that IGF-I treatment of quiescent Swiss 3T3 cells results in the stimulation of nuclear DAG kinase activity. Time course analysis showed an inverse relationship between nuclear DAG mass and DAG kinase activity levels. After IGF-I treatment, maximal enhancement of DAG kinase activity was measured in the internal matrix domain of the nucleus. PKC-alpha remained within the nuclear compartment, even when nuclear DAG mass returned to basal levels. This was conceivably due to interactions with specific nuclear PKC-binding proteins, some of which were identified as lamins A, B, and C and protein C23/nucleolin. Treatment of cells with two DAG kinase inhibitors, R59022 and R59949, blocked the IGF-I-dependent rise in nuclear DAG kinase activity and maintained elevated intranuclear levels of DAG. The two inhibitors also markedly potentiated the mitogenic effect of IGF-I. These results suggest that nuclear DAG kinase plays a key role in regulating the levels of DAG present in the nucleus and that DAG is a key molecule for the mitogenic effect that IGF-I exerts on Swiss 3T3 cells.     

Effect of hyperthermia and protein kinase C inhibitors on DNA synthesis and cell proliferation on Ehrlich ascites tumour cells in vitro.

Effect of hyperthermia and/or protein kinase inhibitors on DNA synthesis and cell proliferation was investigated in Ehrlich ascites tumour cells in vitro. Both H-7 and H-8, potent inhibitors of protein kinase C, suppressed DNA synthesis significantly, but HA1004, an inhibitor of cAMP- and cGMP-dependent protein kinase, did not. Hyperthermia increased greatly the suppressive activity of H-7 and H-8 but not that of HA1004. H-7 also inhibited cell growth. These results suggest that the inhibition of protein kinase C enhances the suppression of DNA synthesis and the proliferation of tumour cells by hyperthermia.     

Role of protein kinase C in phosphorylation of vinculin in adriamycin-resistant HL-60 leukemia cells

In response to phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), HL-60 cells differentiate to macrophage-like cells and exhibit the ability to phosphorylate vinculin in vitro. Adriamycin-resistant HL-60 (HL-60/ADR) cells similarly demonstrate this characteristic without prior treatment with TPA. Since protein kinase C (PK-C) is a cellular TPA receptor, we have examined the role of this enzyme in the inherent ability of HL-60/ADR cells to phosphorylate vinculin. DEAE-cellulose chromatography of cell extracts revealed that HL-60/ADR cells contained 2-fold more PK-C than did the parental cell line. All PK-C activity was found in the cytosol of wild type HL-60 cells, whereas 85% of PK-C activity was cytosolic and 15% was membrane-bound in HL-60/ADR cells. After a 2-day treatment with 10 nM TPA, PK-C activity was reduced 80-90% in both cell lines regardless of its intracellular distribution. Immunoblotting of cell extracts from HL-60/ADR cells or HL-60 cells following treatment with TPA revealed increased levels of a 52-kDa species of similar mass to M-kinase. Coincident with these changes after TPA treatment was a reduction in Ca2+ and phospholipid-independent phosphorylation of vinculin in vitro in extracts from HL-60/ADR cells, whereas HL-60 cells exhibited an elevation of this phosphoprotein. The phosphorylation of vinculin in TPA-treated HL-60 cells or untreated HL-60/ADR cells was blocked by antibodies to protein kinase C. These results suggest that it is not the absolute level of protein kinase C but rather the proteolytic activation of PK-C to a Ca2+ and phospholipid-independent form which is associated with the utilization of vinculin as an endogenous substrate.     

Enhancement of sensitivity to CDDP in human pharyngeal carcinoma cells by H-7, a protein kinase inhibitor

We investigated the synergetic effects of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) on cis-diamminedichloroplatinum(II) (CDDP) cytotoxicity to KB cells, a human pharyngeal carcinoma cell line, and explored the possible mechanism whereby it exerted these effects. In vitro treatment with H-7 markedly decreased protein kinase C (PKC) activity in KB cells and significantly sensitized the cells to CDDP. Treatment of KB cells with H-7 induced a dose-dependent depletion of intracellular glutathione (GSH) levels. These results suggest that the decreased GSH content induced by H-7 may be involved in sensitizing KB cells to CDDP.     

Invasive and metastatic potential of a v-Ha-ras-transformed human bronchial epithelial cell line

The in vivo growth behavior and invasive potential of normal and "immortalized" human bronchial epithelial cells were studied by xenotransplantation procedures, an in vitro assay of invasiveness, and determinations of type IV collagenase activity and mRNA expression. BEAS-2B cells, immortalized after hybrid virus infection (adenovirus 12-simian virus 40), reconstituted a columnar epithelium when xenotransplanted into de-epithelialized rat tracheas transplanted sc into athymic BALB/c mice. A few adenomatous growths could be seen 16 weeks after transplantation. BZR cells, obtained by transfer of the v-Ha-ras oncogene into BEAS-2B cells, were tumorigenic in this xenotransplantation model. BZR-T33 cells, obtained from a tumor produced after injection of BZR cells, were also tumorigenic; however, they exhibited a shorter latent period. When these same cell lines were injected sc and iv into athymic BALB/c mice, BEAS-2B cells were not tumorigenic, and the BZR-T33 cells were more tumorigenic than the BZR cells. The incidence of spontaneous metastases after sc inoculation was zero for BEAS-2B cells, 33% for BZR cells, and 100% for BZR-T33 cells. Similar increasing values that correlated well with the data on in vivo growth were noted in the in vitro invasion assay, the collagenolytic ability, and the mRNA expression of type IV collagenase. Normal human bronchial epithelial cells showed the lowest values in all the assays. These progressive changes occurring in cells derived from the same parental line indicate that the presence of the v-Ha-ras oncogene in immortalized bronchial cells is associated with a full-fledged malignant phenotype, which is further enhanced by in vivo passaging.     

Tumor progression of murine epidermal cells after treatment in vitro with 12-O-tetradecanoylphorbol-13-acetate or retinoic acid

Tumor-promoting or antipromoting agents potentially may act directly on initiated squamous epithelial cells or indirectly through effects on normal keratinocytes or immune cells. The purpose of this study was to examine direct effects by comparing in vitro and in vivo treatment of initiated cell populations with 12-O-tetradecanoylphorbol-13-acetate (TPA) or retinoic acid. Keratinocytes were initiated by treatment in vitro with 7,12-dimethylbenz[alpha]anthracene. Replicate cultures of a cloned initiated cell line were exposed to TPA or retinoic acid with acetone as control. After an equivalent number of population doublings, cultured cell sheets were transplanted as skin grafts to athymic nude mice. Replicate grafts from each in vitro treatment group were then treated with TPA or retinoic acid for 8 months. Promotion was quantified by tumor incidence (graft sites with tumor per total sites) and by tumor growth rate. The findings were as follows: (a) TPA increased tumor incidence whether it was applied in vitro or in vivo; (b) TPA in vitro favored more progressive tumors than TPA in vivo; (c) stages of malignant progression from cloned keratinocytes treated in vitro were histologically identical to those following treatment of skin in vivo, including papilloma, dysplastic invasive papilloma, squamous cell carcinoma, and metastasis to lymph node and lung; (d) retinoic acid treatment in vivo reduced tumor incidence and tumor growth rate in initiated cells previously exposed to TPA but not in cells previously exposed to retinoic acid. The results indicated the following: (a) direct effects of TPA on initiated keratinocyte populations were a significant component of tumor promotion; (b) factors in vivo modified the TPA response toward less progressive growth; and (c) the effect of retinoic acid was modulated by prior treatment history.     

The in vitro effects of H-89, a specific inhibitor of protein kinase A, in the human colonic carcinoma cell line Caco-2.

SUMMARY: H-89 is a compound characterized in vitro as a potent and selective inhibitor of protein kinase A. In the present study, we observed that H-89 induced morphological transformation and caused growth inhibition of the human colon cancer cell line Caco-2 in a dose-dependent manner. However, another protein kinase A inhibitor, H-8, had no effect on Caco-2 cells. To evaluate the possible molecular mechanism of H-89-evoked effects in Caco-2 cells, we analysed the capacity of H-89 to regulate the protein kinase B (Akt/PKB) signalling pathway. H-89 treatment led to an activation of Akt/PKB in Caco-2 cells. This activation was phosphatidylinositol 3 (PI3)-kinase-dependent and promoted survival of Caco-2 cells because the PI3 kinase inhibitor LY294002 inhibited the Akt/PKB activation and induced apoptosis of Caco-2 cells. To test whether Akt/PKB activity promoted resistance to H-89-induced effects, LY294002 was added in combination with H-89. LY294002 greatly potentiated the H-89-induced growth inhibition and apoptosis of Caco-2 cells. These results suggest that the H-89-induced growth inhibition of Caco-2 cells is associated with phosphorylation of Akt/PKB protein and that the cells become more sensitive to H-89 and die by apoptosis upon inhibition of the PI3K/Akt pathway.     

Cell-to-cell communication: a differential response to TGF-beta in normal and transformed (BEAS-2B) human bronchial epithelial cells

The effects of transforming growth factor beta (TGF-beta) on cell-to-cell communication were investigated in the log phase of growth in normal BE and in adenovirus-12 SV40 hybrid virus transformed BE cells (strain BEAS-2B). Gap junctions in these cells were identified immunocytochemically. Exposure of BE cells to exogenous TGF-beta (0.04-4.0 pM) in serum-free keratinocyte growth medium (KGM) for 1 or 24 h reduced the rate of fluorescent dye transfer (i.e. cell-to-cell communication) by 30-50% in BE cells. Inversely, in BEAS-2B cells, TGF-beta after 1 h induced a 2- to 10-fold increase in the rate of dye transfer. After 24 h of TGF-beta, communication among BEAS-2B cells was not significantly different from controls (no exogenous TGF-beta). The protein kinase C (PKC) inhibitor H-7 induced a dose-dependent enhancement in communication, which was even higher in the presence of TGF-beta (4 pM X 24 h). The calmodulin antagonist W-7 enhanced communication in BEAS-2B cells independently of the presence of TGF-beta. In keratinocyte basal medium (KBM) supplemented with EGF (5 ng/ml) or with TGF-beta (4.0 pM) dye transfer was reduced or enhanced respectively. The combination of EGF and TGF-beta in KBM antagonized the stimulatory effect of the latter on communication in BEAS-2B cells. In BE cells, continuous exposure (4 days) to TGF-beta in KGM induced a dose-dependent inhibition of proliferation and an increased expression of a keratinized, epidermoid phenotype. This correlated with a reduction in the expression of a mucous secretory phenotype. Increased exposure to TGF-beta (0.04-4.0 pM) decreased the labeling index in BEAS-2B cells, but the cells retained a growth advantage over normal BE cells, and did not express a keratinized epidermoid morphology. With respect to dye transfer as an index of cell-to-cell communication, we conclude (i) that an inhibition or enhancement of communication is involved in the response of bronchial epithelial cells to mitogens (e.g. epidermal growth factor) or growth inhibitors (e.g. TGF-beta), (ii) that PKC and Ca(2+)-calmodulin-dependent processes regulate dye transfer, and (iii) the effects of TGF-beta are mediated by PKC.     

Dissimilar effects of phorbol ester and diacylglycerol derivative on protein kinase activity in the monoblastoid U937 cell

Mechanism, in addition to protein kinase C activation may mediate 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated differentiation of leukemic cells. We compared the effect of pretreating intact monoblastoid U937 cells with TPA or the diacylglycerol derivative, 1-oleoyl-2-acetylglycerol (OAG), by studying the protein kinase C dependent and independent histone phosphotransferase activity, the phosphorylation of endogenous substrates, and the ability to stimulate differentiation. In cellular fractions derived from cells treated with TPA or OAG, cytosolic protein kinase C activity decreased. In the detergent extracted particulate fraction, TPA produced a time and dose dependent decrease in protein kinase C activity. In contrast, OAG increased particulate protein kinase C activity. In addition, the particulate fraction derived from cells treated with TPA exhibited increased phosphatidyl serine and diolein independent histone phosphotransferase activity as well as an increase in the phosphorylation of two endogenous substrates with molecular weights of 120,000 and 80,000. OAG did not mimic these effects. When exposed to 32P-labeled intact cells, OAG and TPA stimulated phosphorylation of three substrates. Thus, the inability of OAG to mimic the effects of TPA was not due to lack of protein kinase C activation. TPA, but not OAG, stimulated differentiation of the U937 cell to a monocyte-like cell. These data demonstrate that TPA and OAG have dissimilar effects on protein kinase activity and differentiation in the U937 monoblastoid cell.     

Differential regulation of vimentin mRNA by 12-O-tetradecanoylphorbol 13-acetate and all-trans-retinoic acid correlates with motility of Hep 3B human hepatocellular carcinoma cells

Vimentin is a growth-related gene and often expressed when epithelial cells are stimulated to proliferate by growth factors. In cancer, vimentin expression is associated with a dedifferentiated malignant phenotype, increased motility, invasive ability and poor prognosis. We studied the regulation of vimentin mRNA and multistep invasion processes following treatment of 12-O-tetradecanoylphorbol 13-acetate (TPA) and all-trans-retinoic acid (RA) in Hep 3B hepatocellular carcinoma cells. TPA showed marked induction of vimentin mRNA, while RA decreased the mRNA level. TPA or RA did not affect cell proliferation, cell-matrix protein adhesion, and matrix metalloproteinases and urokinase plasminogen activator activities. In vitro invasion ability was significantly increased or decreased with TPA or RA treatment, paralleled to the in vitro motile activity, respectively. These findings suggest that TPA and RA could modulate the invasive potential of Hep 3B cells by altering cellular motility related to differential regulation of vimentin mRNA.     

Annexin V inhibits the 12-O-tetradecanoylphorbol-13-acetate-induced activation of Ras/extracellular signal-regulated kinase (ERK) signaling pathway upstream of Shc in MCF-7 cells

Annexin V is a Ca2+-dependent phospholipid binding protein. Although it has been shown to inhibit protein kinase C (PKC) in cell-free systems, its role in the intact cell is unclear. A stable MCF-7 human breast cancer cell overexpression system was established to investigate the function of annexin V. In these cells, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation and kinase activity of ERK1/2 were suppressed. Morphological changes induced by TPA were reduced by annexin V overexpression as well as by the pan-PKC inhibitor, bisindolylmaleimide I, and by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor, PD98059. TPA-induced MEK1/2 and Raf-1 phosphorylation were reduced in these cells. The TPA-enhanced active Ras, and its association with Raf-1, were reduced. TPA treatment of MCF-7 cells caused an increased association of Shc with Grb2. However, this increased association was prevented in the annexin V-overexpressors. p21WAF/CIP1 is responsible for inhibition of cell cycle progression in MCF-7 cells. TPA induced the expression of p21WAF/CIP1 to a greater extent in MCF-7 parent and control plasmid cells than in annexin V overexpressors. PD98059 inhibited this increase, suggesting that TPA upregulation of p21WAF/CIP1 occurs via the MEK pathway, and that annexin V overexpression blunts it. This work shows that annexin V overexpression suppresses the TPA-induced Ras/ERK signaling by inhibiting at/or upstream of Shc, possibly through the inhibition of PKCs. Oncogene (2000).     

Induction by some protein kinase inhibitors of differentiation of a mouse megakaryoblastic cell line established by coinfection with Abelson murine leukemia virus and recombinant SV40 retrovirus.

Mouse C1 line cells are megakaryoblastic cells established by coinfection of Abelson murine leukemia virus and recombinant simian virus 40. We examined the effects of various compounds on growth and differentiation of these cells. Megakaryocytic differentiation of C1 cells was not induced by cytokines that stimulate megakaryocytic maturation of normal progenitor cells, such as interleukin 3 and 6 and granulocyte-macrophage colony-stimulating factor. However, the cells were induced to differentiate into megakaryocytes by treatment with some protein kinase inhibitors. The inhibition of v-abl tyrosine kinase activity preceded induction of differentiation of the cells treated with tyrosine kinase inhibitors such as genistein, herbimycin A, and erbstatin. Treatment of C1 cells with a v-abl antisense oligomer inhibited their proliferation and induced acetylcholinesterase activity, a typical marker of megakaryocytic differentiation. These results suggest that inhibition of v-abl function is associated with induction of megakaryocytic differentiation of C1 cells. Among the compounds tested, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of cyclic nucleotide-dependent and Ca(2+)-phospholipid-dependent (protein kinase C) protein kinases, was the most potent inducer of differentiation of C1 cells. However, the differentiation-inducing effect of H-7 was unlikely to be mediated through inhibition of protein kinase C or cyclic nucleotide-dependent kinases, because other types of inhibitors of these kinases were not effective, and a protein kinase activator (phorbol ester) induced differentiation of C1 cells. Moreover, neither v-abl mRNA expression nor v-abl kinase activity in C1 cells was affected by treatment with H-7. These findings indicate that induction of megakaryocytic differentiation by H-7 is not related to inhibition of v-abl kinase, but rather to some novel function of H-7.     

Breast cancer cell invasiveness: Correlation with protein kinase C activity and differential regulation by phorbol ester in estrogen receptor-positive and -negative cells

Increased protein kinase C (PKC) activity in malignant breast tissue and in most aggressive breast cancer cell lines has suggested a possible role of PKC in breast carcinogenesis and tumor progression. We have investigated here the involvement of PKC in the in vitro invasiveness and motility of several breast cancer cell lines. Modulation of PKC activity by treatment with a phorbol ester (TPA), drastically increased the invasiveness of 2 estrogen receptor-positive (ER⁺) lines (MCF7 and ZR 75.1), whereas it markedly decreased the invasiveness of 2 ER⁻ cell lines (MDA-MB-231 and MDA-MB-435). A PKC inhibitor (H7) reversed the TPA effects in MCF7 cells, whereas it mimicked TPA action in MDA-MB-231 cells. All of these effects of TPA also were observed to a similar extent for cell chemotaxis, and they were not dependent on protein neo-synthesis. In parallel, short TPA treatment induced cell spreading and microtubule organization in MCF7 cells and inverse morphological changes in MDA-MB-231 cells. In ER⁺ cells, constitutive PKC activity and PKCα expression were very low as compared to ER⁻ cells, and this correlated with the invasive potential of the cells. The opposed effects of TPA in ER⁺ and ER⁻ cells could be due to the abnormal TPA regulation of PKCα observed in ER⁻ cells. Int. J. Cancer 75:750–756, 1998.© 1998 Wiley-Liss, Inc.     

Effect of hyperthermia and protein kinase C inhibitors on DNA synthesis and cell proliferation on Ehrlich ascites tumour cells in vitro

Effect of hyperthermia and/or protein kinase inhibitors on DNA synthesis and cell proliferation was investigated in Ehrlich ascites tumour cells in vitro. Both H-7 and H-8, potent inhibitors of protein kinase C, suppressed DNA synthesis significantly, but HA1004, an inhibitor of cAMP- and cGMP-dependent protein kinase, did not. Hyperthermia increased greatly the suppressive activity of H-7 and H-8 but not that of HA1004. H-7 also inhibited cell growth. These results suggest that the inhibition of protein kinase C enhances the suppression of DNA synthesis and the proliferation of tumour cells by hyperthermia.