Phorbol ester induced MDR1 expression in K562 cells occurs independently of mitogen-activated protein kinase signaling pathways.

The MDR1 gene encoding the multidrug pump P-glycoprotein is transcriptionally activated in response to diverse extracellular stimuli, including the tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the signal transduction pathway responsible is unknown. Downstream of protein kinase C (PKC), the effects of TPA are often mediated by the Raf-1/MEK/ERK mitogen-activated protein kinase (MAPK) cascade, and Raf-1 has been implicated in MDR1 induction by serum and mitogens. Therefore, we examined the potential role of MAPK activation in TPA-mediated MDR1 induction in human leukemia K562 cells. MDR1 mRNA expression was significantly increased by TPA in the concentration range of 4 - 100 nM, with a maximal response 5 - 10 h after TPA addition. TPA-mediated MDR1 induction was inhibited by several PKC inhibitors including staurosporine, H7 and calphostin C. TPA stimulated the subcellular translocation of PKCalpha from the cytosol to the membrane and nucleus but did not affect other PKC isozymes. TPA also activated the Raf1/MEK/ERK cascade and activated another MAPK member, p38, but not JNK. In order to determine the potential role of MAPKs in MDR1 induction by TPA, specific inhibitors were utilized. The MEK inhibitor PD 098059, as well as the PKC inhibitors, completely blocked TPA-mediated ERK activation. However, under identical conditions, MDR1 induction by TPA was completely unaffected by PD 098059. Furthermore, SB 202190, which effectively inhibited TPA-mediated p38 activation, failed to inhibit TPA-induced MDR1 mRNA expression. These data demonstrate that MDR1 induction by TPA occurs via a PKC-dependent mechanism that operates independently of ERK, p38 or JNK pathways, and thus have important implications for understanding the mechanisms of MDR1 induction by extracellular stimuli.

Role of the stress-activated/c-Jun NH2-terminal protein kinase pathway in the cellular response to adriamycin and other chemotherapeutic drugs.

c-Jun NH2-terminal protein kinase (JNK), a member of the mitogen-activated protein kinase family, is activated in response to many stressful stimuli including heat shock, UV irradiation, protein synthesis inhibitors, and inflammatory cytokines. In this study, we investigated whether JNK plays a role in the cellular response to different drugs commonly used in cancer chemotherapy. Treatment of human KB-3 carcinoma cells with Adriamycin resulted in a time- and dose-dependent activation of JNK of up to 40-fold. Treatment with vinblastine or etoposide (VP-16) also activated JNK, with maximum increases of 6.5- and 4.3-fold, respectively. Consistent with these findings, increased c-Jun phosphorylation was observed after drug treatment of cells. In contrast, none of the drugs significantly activated the extracellular response kinase/mitogen-activated protein kinase pathway. Since these drugs are transport substrates for the MDR1 gene product, P-glycoprotein, JNK was assayed in two multidrug-resistant (MDR) KB cell lines, KB-A1 and KB-V1, selected for resistance to Adriamycin and vinblastine, respectively. Relative to KB-3 cells, basal JNK activity was increased 7-fold in KB-A1 cells and 4-fold in KB-V1 cells, with no change in JNK protein expression, indicating that JNK is present in a more highly activated form in the MDR cell lines. Under conditions optimal for JNK activation, Adriamycin, vinblastine, and VP-16 all induced MDR1 mRNA expression in KB-3 cells. Our findings suggest that JNK activation is an important component of the cellular response to several structurally and functionally distinct anticancer drugs and may also play a role in the MDR phenotype.

Expression, subcellular distribution and response to phorbol esters of protein kinase C (PKC) isozymes in drug-sensitive and multidrug-resistant KB cells evidence for altered regulation of PKC-alpha.

Protein kinase C (PKC) comprises a family of related phospholipid-dependent serine/threonine protein kinases. PKC has been implicated in the induction and maintenance of the multidrug-resistance (MDR) phenotype but the role of different isozymes is not well understood. We compared the expression and subcellular distribution, and membrane association and down-regulation induced by phorbol esters, of individual PKC isozymes in drug-sensitive KB-3 and multidrug-resistant KB-V1 human carcinoma cell lines. Immunoblotting with isozyme-specific antibodies indicated the presence of PKC alpha (cytosol only). PKC beta (membrane only). PKC epsilon (mainly membrane associated) and PKC zeta (both fractions). PKC delta and PKC gamma were not detected. The expression levels of PKC beta. PKC epsilon and PKC zeta were unchanged in KB-V1 cells; PKC alpha was modestly increased ( approximately 65%) in the resistant cells as further determined by enzyme assay. The cytosolic nature and increased expression of PKC alpha were confirmed by immunofluorescent localization studies. Revertant cells, obtained by culturing KB-V1 cells in a drug-free medium, regained drug sensitivity with a loss of P-glycoprotein and a concomitant decrease in expression of PKC alpha, KB-V1 cells were found to differ markedly from KB-3 cells with respect to the translocation and down-regulation specifically of PKC alpha upon exposure to 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA). Treatment with 30 nM TPA for 24 h completely depleted KB-3 cells of PKC alpha whereas 1 microM TPA was required to deplete KB-V1 cells of PKC alpha. Similar results were obtained when phorbol-12, 13-dibutyrate was used instead of TPA. Defective TPA-mediated down-regulation of PKC alpha was also observed in another PKC alpha-overexpressing MDR cell line. KB-A1. Importantly, cellular uptake of radiolabeled phorbol ester was similar for both drug-sensitive and MDR cells. Sensitive and resistant cells exhibited similar expression levels of RACK1, a PKC-binding protein important in activation-induced translocation. These findings further highlight the importance of PKC alpha in the MDR phenotype, and suggest that this isozyme may be expressed in a modified form or be subject to an altered regulation in MDR cells.

SAGE II aerosol validation: selected altitude measurements, including particle micromeasurements.

Correlative aerosol measurements taken at a limited number of altitudes during coordinated field experiments are used to test the validity of particulate extinction coefficients derived from limb path solar radiance measurements taken by the Stratospheric Aerosol and Gas Experiment (SAGE) II Sun photometer. In particular, results are presented from correlative measurement missions that were conducted during January 1985, August 1985, and July 1986. Correlative sensors included impactors, laser spectrometers, and filter samplers aboard an U-2-airplane, an upward pointing lidar aboard a P-3 airplane, and balloon-borne optical particle counters (dustsondes). The main body of this paper focuses on the July 29, 1986, validation experiment, which minimized the many difficulties (e.g., spatial and temporal inhomogeneities, imperfect coincidences) that can complicate the validation process. On this day, correlative aerosol measurements taken at an altitude of 20.5 km agreed with each other within their respective uncertainties, and particulate extinction values calculated at SAGE II wavelengths from these measurements validated corresponding SAGE II values. Additional validation efforts on days when measurement and logistical conditions were much less favorable for validation are discussed in an appendix.

SAGE II aerosol data validation based on retrieved aerosol model size distribution from SAGE II aerosol measurements.

This paper describes an investigation of the comprehensive aerosol correlative measurement experiments conducted between November 1984 and July 1986 for satellite measurement program of the Stratospheric Aerosol and Gas Experiment (SAGE II). The correlative sensors involved in the experiments consist of the NASA Ames Research Center impactor/laser probe, the University of Wyoming dustsonde, and the NASA Langley Research Center airborne 14-inch (36 cm) lidar system. The approach of the analysis is to compare the primary aerosol quantities measured by the ground-based instruments with the calculated ones based on the aerosol size distributions retrieved from the SAGE II aerosol extinction measurements. The analysis shows that the aerosol size distributions derived from the SAGE II observations agree qualitatively with the in situ measurements made by the impactor/laser probe. The SAGE II-derived vertical distributions of the ratio N0.15/N0.25 (where Nr is the cumulative aerosol concentration for particle radii greater than r, in micrometers) and the aerosol backscatter profiles at 0.532- and 0.6943-micrometer lidar wavelengths are shown to agree with the dustsonde and the 14-inch (36-cm) lidar observations, with the differences being within the respective uncertainties of the SAGE II and the other instruments.