Response of malignant glioma cell lines to activation and inhibition of protein kinase C-mediated pathways

1990 ◽  
Vol 73 (1) ◽  
pp. 98-105 ◽  
Author(s):  
Ian F. Pollack ◽  
Margaret S. Randall ◽  
Matthew P. Kristofik ◽  
Robert H. Kelly ◽  
Robert G. Selker ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Malignant Glioma ◽  
Polymyxin B ◽  
Content Type ◽  
Rat Glioma ◽  
Kinase C ◽  
Low Passage

✓ To evaluate the role of protein kinase C-mediated pathways in the proliferation of malignant gliomas, this study examined the effect of a protein kinase C (PKC)-activating phorbol ester (12-O-tetradecanoyl-13-phorbol acetate or TPA) and a protein kinase C inhibitor (polymyxin B) on deoxyribonucleic acid (DNA) synthesis of malignant glioma cells in vitro. A serum-free chemically defined medium, MCDB 105, was employed for all studies. Two established human malignant glioma cell lines (T98G and U138), two rat glioma lines (9L and C6), and two low-passage human glioma lines (obtained from surgical specimens) were studied. With the exception of the C6 line, all tumors responded in a dose-dependent fashion to nanomolar concentrations of TPA with a median effective dose that varied from 0.5 ng/ml for the U138 glioma to 1 ng/ml for the T98G glioma. At optimal concentrations (5 to 10 ng/ml), TPA produced a two- to five-fold increase in the rate of DNA synthesis (p < 0.05) as assessed by incorporation of 3H-thymidine. However, TPA had no additive effect on the mitogenic response produced by epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). Inhibition of PKC using the antibiotic polymyxin B (20 µg/ml) abolished the TPA-induced mitogenic response in the five responsive lines tested. In two tumors (U138 and 9L), polymyxin B also eliminated EGF-, PDGF-, and serum-induced DNA synthesis as well as abolishing baseline DNA synthesis. These cells remained viable, however, as assessed by trypan blue exclusion; after removal of polymyxin B from the medium, they were able to resume DNA synthesis in response to TPA and serum. In the three other tumors (T98G and the two low-passage human glioma lines), growth factor-induced and serum-induced DNA synthesis were inhibited by approximately 25% to 85%. It is concluded that PKC-mediated pathways affect DNA synthesis in the human malignant glial tumors studied. The response of the glioma cells to TPA is similar to the responses seen in fetal astrocytes, but differs significantly from those reported for normal adult glial cultures. Because the response of the 9L glioma to TPA is similar to the responses seen in the human tumors, the 9L rat glioma model may prove useful for examining the role of PKC-mediated pathways in controlling glioma growth in vivo.

Involvement of PDGF in pressure-induced mesangial cell proliferation through PKC and tyrosine kinase pathways

1999 ◽  
Vol 277 (1) ◽  
pp. F105-F112 ◽  
Author(s):  
Hiroaki Kato ◽  
Akihiko Osajima ◽  
Yasuhito Uezono ◽  
Masahiro Okazaki ◽  
Yuki Tsuda ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Dna Synthesis ◽  
Tyrosine Kinases ◽  
Mechanical Stretch ◽  
Transmural Pressure ◽  
Kinase C

In glomerular hypertension, mesangial cells (MC) are subjected to at least two physical forces: mechanical stretch and high transmural pressure. Increased transmural pressure, as well as mechanical stretch, promotes MC proliferation, which may enhance glomerulosclerosis. The exact mechanism of this effect is not fully understood. We examined the effects of transmural pressure alone on cell proliferation and DNA synthesis and investigated the role of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), candidates for mediation of glomerular diseases, in the pressure-induced events. Pressure was applied to cultured MC placed in a sealed chamber using compressed helium gas. Application of pressure resulted in a time-dependent (∼2 h) and pressure level-dependent (∼80 mmHg) increase in cell number (1.4-fold) and [3H]thymidine incorporation (2.7-fold). Pressure-induced DNA synthesis was significantly suppressed by inhibitors of phospholipase C (2-nitro-4-carboxyphenyl- N, N-diphenylcarbamate), protein kinase C [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and chelerythrine], or tyrosine kinases (genistein). Pressure caused a rapid but transient formation of inositol 1,4,5-trisphosphate, which was blocked by the phospholipase C inhibitor. Pressure also promoted a rapid increase in tyrosine kinase activity. Pressure increased mRNA levels of PDGF-B, with a peak at 6 h, but not those of PDGF-A or bFGF. Pressure-induced DNA synthesis was partially inhibited by a neutralizing anti-PDGF antibody but not by an antibody against bFGF or nonimmune IgG. Our results indicated that pressure by itself increases DNA synthesis and proliferation of cultured rat MC possibly through activation of protein kinase C and tyrosine kinases, and PDGF-B could be partially involved in these pathways.

Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells

1990 ◽  
Vol 10 (10) ◽  
pp. 5314-5323
Author(s):  
H Cai ◽  
J Szeberényi ◽  
G M Cooper
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
3T3 Cells ◽  
Kinase C ◽  
Mitogenic Signal Transduction ◽  
Nih 3T3 ◽  
Mitogenic Signal

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.

Endothelin-1 stimulates DNA synthesis and proliferation of pulmonary artery smooth muscle cells

1992 ◽  
Vol 263 (6) ◽  
pp. C1295-C1301 ◽  
Author(s):  
K. Janakidevi ◽  
M. A. Fisher ◽  
P. J. Del Vecchio ◽  
C. Tiruppathi ◽  
J. Figge ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Pulmonary Artery ◽  
Dna Synthesis ◽  
Synthetic Activity ◽  
Proliferative Potential ◽  
Endothelin 1 ◽  
Kinase C

Endothelin-1 (ET-1), a 21-amino acid peptide released from the endothelium, elicits a variety of biological effects that include vascular smooth muscle cell (VSMC) contraction, release of secondary mediators, and cell proliferation. The present study was undertaken to examine the proliferative potential of ET-1 toward pulmonary artery VSMC in culture. In the presence of low serum and epidermal growth factor (EGF), ET-1 stimulated marked DNA synthesis and proliferation of VSMC. The contributing factor from serum appeared to be platelet-derived growth factor (PDGF) because the antibody to PDGF eliminated the stimulatory activity. The antibody to EGF also prevented the stimulation, suggesting that both PDGF and EGF are required for the full expression of the VSMC growth-promoting activity of ET-1. A paradoxical aspect of ET-1 effect on VSMC was the ability of ET-1 to inhibit the EGF-stimulated DNA synthesis when the two factors were added together to a high baseline DNA synthetic activity. The inhibition was prevented if ET-1 was added 12-18 h after the addition of EGF or if ET-1 and EGF were added to a protein kinase C-depleted VSMC. The inhibition by ET-1 may be mediated by protein kinase C activation followed by inhibition of EGF binding to its receptor. The results indicate that ET-1 under appropriate conditions can modulate the growth of pulmonary artery VSMC in both positive and negative directions.

Growth factor-induced DNA synthesis in cells that overproduce protein kinase C

1990 ◽  
Vol 145 (2) ◽  
pp. 262-267 ◽  
Author(s):  
Sadayori Hoshina ◽  
Marius Ueffing ◽  
I. Bernard Weinstein
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Kinase C ◽  
In Cells

scholarly journals Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells.

1990 ◽  
Vol 10 (10) ◽  
pp. 5314-5323 ◽  
Author(s):  
H Cai ◽  
J Szeberényi ◽  
G M Cooper
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
3T3 Cells ◽  
Kinase C ◽  
Mitogenic Signal Transduction ◽  
Nih 3T3 ◽  
Mitogenic Signal

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.

scholarly journals Mitogenic signaling pathways of growth factors can be distinguished by the involvement of pertussis toxin-sensitive guanosine triphosphate-binding protein and of protein kinase C.

1990 ◽  
Vol 1 (10) ◽  
pp. 747-761 ◽  
Author(s):  
N Nishizawa ◽  
Y Okano ◽  
Y Chatani ◽  
F Amano ◽  
E Tanaka ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Growth Factors ◽  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Signaling Pathways ◽  
Pertussis Toxin ◽  
Guanosine Triphosphate ◽  
Kinase C

We have examined the possible involvements of pertussis toxin (PT)-sensitive guanosine triphosphate (GTP)-binding protein (Gp) and protein kinase C (PKC) in the mitogenic signaling pathways of various growth factors by the use of PT-pretreated and/or 12-O-tetradecanoyl phorbol-13-acetate (TPA)-pretreated mouse fibroblasts. Effects of PT pretreatment (inactivation of PT-sensitive Gp) and TPA pretreatment (depletion of PKC) on mitogen-induced DNA synthesis varied significantly and systematically in response to growth factors: mitogenic responses of cells to thrombin, bombesin, and bradykinin were almost completely abolished both in PT- and TPA-pretreated cells; responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vanadate were reduced to approximately 50% both in PT- and TPA-pretreated cells compared with native cells; response to basic fibroblast growth factor (bFGF) was not affected in PT-pretreated cells but was inhibited to some extent in TPA-pretreated cells. Thus, growth factors examined have been classified into three groups with regard to the involvements of PT-sensitive Gp and PKC in their signal transduction pathways. Binding of each growth factor to its receptor was not affected significantly by pretreatment of cells with PT or TPA. Inhibitory effects of PT and TPA pretreatment on each mitogen-induced DNA synthesis were not additive, suggesting that the functions of PT-sensitive Gp and PKC lie on an identical signal transduction pathway. Although all three groups of mitogens activated PKC, signaling of each growth factor depends to a varying extent on the function of PKC. Our results indicate that a single peptide growth factor such as EGF, PDGF, or bFGF acts through multiple signaling pathways to induce cell proliferation.

Phorbol esters activate protein kinase C and glucose transport and can replace the requirement for growth factor in interleukin-3-dependent multipotent stem cells

1986 ◽  
Vol 84 (1) ◽  
pp. 93-104 ◽  
Author(s):  
A.D. Whetton ◽  
C.M. Heyworth ◽  
T.M. Dexter
Keyword(s):  
Stem Cells ◽  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Dna Synthesis ◽  
Glucose Transport ◽  
Hexose Transport ◽  
Cellular Survival ◽  
Interleukin 3 ◽  
Kinase C

Interleukin 3 (IL-3) promotes the survival, proliferation and development of progenitor cells from several distinct haemopoietic lineages and can also stimulate the self-renewal of stem cells. We have explored the mode of action of this growth factor in promoting survival and proliferation, using a multipotent haemopoietic stem cell line FDC-Mix 1. In the absence of IL-3 these cells died within 16–48 h. However, this requirement for IL-3 could be replaced by 12-O-tetradecanoylphorbol-13-acetate (TPA) plus Ca2+ ionophore, which promoted not only survival but also DNA synthesis with no concomitant loss of the multipotential nature of these cells. TPA and Ca2+ ionophore, respectively, could also interact synergistically with IL-3 to promote DNA synthesis. Both IL-3 and TPA stimulated the translocation of protein kinase C (PK-C) from the cytosol to a membrane-bound form in FDC-Mix 1 cells. Previously we suggested that IL-3 can activate the primary metabolism of IL-3-dependent cells so that increased glucose transport and glycolysis lead to maintenance of ATP levels and cellular survival. To investigate whether TPA and, or, Ca2+ ionophore could also influence cellular survival via an activation of glucose uptake we assessed the effects of these agents on hexose transport. TPA +/− Ca2+ ionophore activated hexose transport to the same degree as does IL-3 but these agents cannot superstimulate FDC-Mix 1 hexose transport in cells that already exhibit an activated transport system from preincubation with IL-3. We conclude that IL-3 maintains FDC-Mix 1 cells via its ability to activate PK-C and increase cytosolic levels of Ca2+, and that an IL-3-mediated activation of PK-C may promote cellular survival via its ability to enhance hexose uptake by phosphorylating the glucose transport protein.

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