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Biomedical subjects

S W Johnson

Publications and source records attributed to S W Johnson.

At least 37 records · Page 2Linked to original sources
PubMed1999

Growth of human tumor cells in macroporous microcarriers results in p53-independent, decreased cisplatin sensitivity relative to monolayers.

Multicellular contact has been shown to influence the in vitro sensitivity of cells to drug treatment. We investigated the use of macroporous gelatin microcarriers, CultiSpher-G, as a convenient laboratory system for the molecular analysis of this "contact effect". We determined that human A549 cells can be grown in CultiSphers with growth and cell cycle parameters similar to those of monolayers. In addition, cells in CultiSphers express less p27/kip1, an indicator of cell cycle arrest, than equivalent cells in monolayers. When treated with drugs, A549 cells grown in CultiSphers or monolayers accumulate equivalent amounts of platinum-DNA adducts and similar amounts of doxorubicin. Moreover, A549 and KB-3-1 cells in CultiSphers have significantly decreased sensitivity to cis-platinum(II)diammine dichloride (cisplatin), 4-hydroperoxycyclophosphamide, doxorubicin, and paclitaxel (taxol) compared with cells in monolayers when assayed by clonogenic survival. Cisplatin treatment in monolayers or CultiSphers did not result in apoptotic cell death. In contrast, paclitaxel caused a significant amount of sub-G1 DNA, an indicator of apoptosis, which was diminished when cells were grown in CultiSphers compared with monolayers. When grown in CultiSphers, cells with abrogated p53 function (A549/16E6 and NCI-H1299) were less sensitive to cisplatin than the corresponding monolayer cells, indicating that the decrease in sensitivity is p53 independent. Taken together, the data suggest that CultiSpher-G microcarriers are a useful in vitro system to examine the effects of three-dimensional cell contact on drug sensitivity of human tumor cells.

Antineoplastic Agents↗
PubMed1998

Nitric oxide facilitates N-methyl-D-aspartate-induced burst firing in dopamine neurons from rat midbrain slices.

Dopamine (DA) neurons in the ventral tegmental area and substantia nigra pars compacta were induced to fire in bursts with application of N-methyl-D-aspartate (NMDA, 20 microM) and apamin (100 nM) while recording intracellularly in the rat brain slice. L-Arginine (300 microM), a substrate for nitric oxide (NO) production, increased both the number of spikes per burst and the magnitude of interburst hyperpolarizations. Nitric oxide synthase inhibitors N-nitro-L-arginine methyl ester (L-NAME, 100 microM), N-nitro-L-arginine, and 7-nitroindazole inhibited NMDA-induced burst firing by reducing the number of spikes per burst. Moreover, L-arginine (100 microM) reversed the inhibition of burst firing produced by L-NAME. These findings suggest that NO facilitates NMDA-induced burst firing in DA neurons.

Animals↗
PubMed1998

Sodium pump evokes high density pump currents in rat midbrain dopamine neurons.

1. Patch pipettes contained various concentrations of Na+ ([Na+]pip) in order to record strophanthidin-sensitive currents under voltage clamp in dopamine neurons in slices of rat substantia nigra and ventral tegmental area. 2. When [Na+]pip was 40 mM and the external K+ concentration ([K+]o) was 2.5 mM, strophanthidin (10 microM) evoked 461 +/- 121 pA of inward current. This effect was concentration dependent, with an EC50 of 7.1 +/- 2.6 microM. At potentials of -60 to -120 mV, strophanthidin-induced currents were not associated with significant changes in chord conductance. 3. Strophanthidin (10 microM) evoked 234 +/- 43 pA of inward current when [Na+]pip was 0.6 mM, and 513 +/- 77 pA when [Na+]pip was 80 mM. Despite higher pump currents with greater [Na+]pip, the strophanthidin EC50 was not significantly different for any of six different [Na+]pip. 4. Sodium pump currents were half-maximal when the [Na+]pip was about 1.3 mM. Maximum pump current was estimated at 830 pA (29 microA cm-2) at concentrations of intracellular Na+ that were assumed to be saturating (50-100 mM). 5. Strophanthidin currents were smaller in a reduced [K+]o (EC50 = 0.2 mM). 6. These data show that intracellular Na+ loading evokes relatively large pump currents. Our results are consistent with the physiological role of the sodium pump in burst firing in midbrain dopamine neurons

Animals↗
PubMed1998

Translocation and activation of AKT2 in response to stimulation by insulin.

The AKT2 oncogene encodes a protein-serine/threonine kinase that was recently shown to be activated by a variety of growth factors. In addition, we previously showed that AKT2 is abundant in brown fat and skeletal muscle, tissues that are highly insulin responsive and that play a role in glucose metabolism. In this study, we demonstrate that AKT2 is activated in response to stimulation by insulin in a dose- and time-dependent manner in human ovarian carcinoma cells and that activation of AKT2 is abolished in cells pretreated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). Activation of AKT2 is manifested by changes in its phosphorylation state. Immunofluorescence experiments demonstrate that AKT2 is translocated to the plasma membrane after insulin stimulation, and this translocation is abolished by wortmannin. Both wild-type AKT2 activated by insulin and constitutively active AKT2, which has been targeted to the membrane by the addition of a myristoylation signal, were found to inactivate glycogen synthase kinase-3 (GSK-3) in vitro. GSK-3 was not inactivated by a catalytically inactive AKT2 mutant. Collectively, these data indicate that activation of AKT2 by insulin is mediated by PI 3-kinase and that GSK-3 is a downstream target of AKT2, suggesting a potentially important role of AKT2 in glycogen synthesis and other GSK-3 signaling pathways.

Androstadienes↗
PubMed1998

Recent insights into platinum drug resistance in cancer.

Cisplatin and its analogs have become important components of chemotherapeutic regimens for the treatment of solid tumors, however, their overall effectiveness is limited by the emergence of drug-resistant tumor cells. Resistance to the platinum drugs is multifactorial consisting of mechanisms that prevent the formation of lethal platinum-DNA adducts and mechanisms that operate downstream of the drug/target interaction to promote cell survival. Continued progress in the study of the drug resistance phenotype as well as the development of new platinum analogs may eventually lead to improved therapies and increased survival rates.

Journal Article↗
PubMed1998

The biology of ovarian cancer.

The biology of ovarian cancer broadly defined covers essentially all aspects of the disease from how it arises to how it responds to chemotherapy, often becomes refractory to treatment, and ultimately kills the patient. In this article, we take the liberty of discussing many of these issues to some degree in context of the "natural/clinical" history/biology of the disease. We focus on concepts of how the disease develops, efforts to identify histologic changes that may precede the development of overt ovarian cancer, efforts to define how the growth and function of the normal ovarian surface epithelium are regulated to gain insights into how aberrant function of these pathways may contribute to the initiation of the disease, molecular biological studies on clinical ovarian cancer specimens, efforts to experimentally induce the malignant transformation of ovarian surface epithelial cells, and efforts to understand why ovarian cancer is often initially responsive to chemotherapy but ultimately becomes refractory.

Cell Transformation, Neoplastic↗
PubMed1998

Gamma-hydroxybutyrate is a GABAB receptor agonist that increases a potassium conductance in rat ventral tegmental dopamine neurons.

gamma-Hydroxybutyric acid (GHB) is an abused substance that occurs naturally in the basal ganglia. Electrophysiological recordings of membrane voltage and current were made to characterize the effects of GHB on dopamine neurons in the ventral tegmental area of the rat midbrain slice. Perfusate containing GHB caused a concentration-dependent membrane hyperpolarization (EC50 = 0.88 +/- 0.21 mM) and a reduction in input resistance (EC50 = 0.74 +/- 0.21 mM). The highest concentration of GHB studied (10 mM) hyperpolarized neurons by 20 +/- 3 mV and reduced input resistance by 58% +/- 9%. Changes in membrane potential and input resistance were blocked by the gamma-aminobutyric acid antagonist CGP-35348 (300 microM), but neither bicuculline (30 microM) nor strychnine (10 microM) was an effective antagonist. Voltage-clamp recordings demonstrated that GHB (1 mM) evoked 80 +/- 6 pA of outward current (at -60 mV) that reversed at -110 mV (in 2.5 mM K+). Increasing concentrations of extracellular K+ progressively shifted the reversal to more depolarized potentials. In tetrodotoxin (0.3 microM) and tetraethylammonium (10 mM), depolarizing voltage steps (to -30 mV) evoked calcium-dependent current spikes that were completely blocked by GHB (1 mM). These data suggest that GHB is an agonist at gamma-aminobutyric acid receptors and would be expected to inhibit DA release by causing K+-dependent membrane hyperpolarization.

Animals↗
PubMed1997

Presynaptic GABAB and adenosine A1 receptors regulate synaptic transmission to rat substantia nigra reticulata neurones.

1. Patch pipettes were used to record whole-cell currents under voltage clamp in substantia nigra zona reticulata (SNR) neurones in the rat midbrain slice. Bipolar electrodes evoked synaptic currents mediated by glutamate (EPSCs) and GABAA receptors (IPSCs). 2. Baclofen reduced the amplitude of IPSCs by 48% at its IC50 value of 0.60 microM. The GABAB antagonist CGP 35348 blocked this effect with a Kd value estimated by Schild analysis of 5 microM. 3. Adenosine reduced IPSCs by 48% at its IC50 value of 56 microM. Adenosine agonists reduced IPSCs with the following rank order of potency: CPA (N6-cyclopentyladenosine) > R-PIA (R(-)N6-(2-phenylisopropyl)adenosine) > CHA (N6-cyclohexyladenosine) = NECA (5'-N-ethylcarboxamidoadenosine) > 2-CADO (2-chloroadenosine) > adenosine. Schild analysis yielded a Kd value of 0.4 nM for antagonism of CPA by the adenosine A1 receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine). 4. Both baclofen and adenosine reduced the magnitude of paired-pulse depression of IPSCs, and neither blocked currents evoked by GABA, which was pressure-ejected from micropipettes. 5. Glutamate EPSCs were reduced by baclofen (IC50 = 0.78 microM) and adenosine (IC50 = 57 microM). Schild analysis yielded a Kd value of 11 microM for antagonism of baclofen-induced inhibition of EPSCs by CGP 35348. DPCPX (1 microM) completely blocked the inhibitory effects of adenosine (100 microM) and CPA (100 nM) on EPSCs. Neither adenosine nor baclofen reduced inward currents evoked by glutamate which was pressure-ejected from micropipettes. 6. These results show that presynaptic GABAB and A1 receptors reduce glutamate and GABA release from nerve terminals in the SNR.

Adenosine↗
PubMed1997

Bicuculline methiodide potentiates NMDA-dependent burst firing in rat dopamine neurons by blocking apamin-sensitive Ca2+-activated K+ currents.

Apamin, a bee venom toxin which blocks a Ca2+-dependent K+ current, potentiates N-methyl-D-aspartate (NMDA)-induced burst firing in dopamine neurons. We now report that burst firing is also potentiated by an apamin-like effect of bicuculline methiodide (BMI) at the same concentration (30 microM) which blocks GABA(A) receptors in vitro. Using microelectrodes to record intracellularly from rat dopamine neurons in the midbrain slice, BMI reduced the apamin-sensitive afterhyperpolarization in all cells tested. BMI also mimicked apamin (100 nM) by potentiating burst firing produced by a concentration of NMDA (10 microM) which is too low to evoke burst firing when perfused alone. When recording under voltage-clamp, both BMI and apamin reduced a depolarization-activated outward current which was also sensitive to perfusate containing no-added Ca2+. Although picrotoxin (100 microM) and bicuculline free base (30 microM) blocked the inhibition of firing produced by the GABA(A) agonist isoguvacine (100 microM), neither had apamin-like effects. We conclude that BMI potentiates burst firing by blocking an apamin-sensitive Ca2+-activated K+ current.

Action Potentials↗
PubMed1997

The chemopreventive agent oltipraz stimulates repair of damaged DNA.

Carcinogens may damage DNA either through the production of radicals that cause base modification in situ or through the formation of bulky adducts at relatively nucleophilic sites. Preclinical studies have demonstrated that administration of the dithiolethione oltipraz protects laboratory animals from the development of tumors following subsequent exposure to a variety of carcinogens. This may occur through a mechanism involving the induction of detoxicating gene expression. In some models, oltipraz treatment following carcinogen exposure may also confer protection. To investigate a possible mechanism for this observation, we studied the effects of oltipraz on base excision repair and platinum-DNA damage formation and removal. No effect of oltipraz was observed on base excision repair as determined by an in vitro assay measuring the repair of apurinic/apyrimidinic sites by untreated and oltipraz-treated HT-29 whole-cell extracts. Treatment of HT-29 cells with cisplatin in the absence or presence of 30 and 100 microM oltipraz decreased the accumulation of platinum in DNA. A dose-dependent reduction in DNA platination was also observed in purified DNA treated concurrently with cisplatin and increasing concentrations of oltipraz. When DNA was first platinated and subsequently incubated with oltipraz, no decrease in platinum content in DNA was found. Preincubation of HT-29 cells with oltipraz enhanced the rate of removal of total platinum-DNA adducts and interstrand cross-links. These data support a novel mechanism through which dithiolethiones may protect carcinogen-exposed animals from tumor formation and may expand their potential role in the clinic.

Anticarcinogenic Agents↗
PubMed1997

Increased platinum-DNA damage tolerance is associated with cisplatin resistance and cross-resistance to various chemotherapeutic agents in unrelated human ovarian cancer cell lines.

We have examined a panel of 12 unrelated human ovarian cancer cell lines derived from patients who were either untreated or treated with platinum-based chemotherapy to determine whether a relationship is present between cisplatin sensitivity and: (a) cellular platinum accumulation; (b) glutathione levels; (c) platinum-DNA adduct formation; (d) platinum-DNA adduct removal; and (e) platinum-DNA damage tolerance. Multiple regression and correlation analysis revealed that of these resistance mechanisms, platinum-DNA damage tolerance correlates strongly with cisplatin sensitivity (r = 0.84, P = 0.001), whereas platinum accumulation (r = -0.11), cellular glutathione levels (r = 0.13), and platinum-DNA adduct removal (r = 0.44) correlate insignificantly. The correlation of platinum-DNA damage tolerance to cisplatin sensitivity (IC50s) is derived from the clustering of platinum-DNA adduct formation into three distinct groups spanning a 3-fold range, which is narrow relative to the corresponding 43-fold range in sensitivity. Adduct formation itself is not associated with cisplatin sensitivity (r = -0.38). Strong correlations were also observed between platinum-DNA damage tolerance and sensitivity to Adriamycin (r = 0.80, P = 0.002), paclitaxel (r = 0.87, P = 0.0002), etoposide (r = 0.78, P = 0.003), and mitomycin C (r = 0.73, P = 0.007). These results suggest that the failure of pathways that are involved in recognizing and processing platinum-DNA damage and other types of drug-induced damage that culminate in cell death may result in a broad resistance phenotype.

Antineoplastic Agents↗
PubMed1997

DNA binding activity of the mammalian translation elongation complex: recognition of chromium- and transplatin-damaged DNA.

The elongation factor complex, EF-1H, serves an essential function in protein biosynthesis in eukaryotic cells, although the role of EF-1H in other physiological processes is unknown. In this report, we demonstrate that three components of EF-1H (EF-1 beta, EF-1 delta, EF-1 gamma) bind to DNA modified with chromium (Cr), a potent DNA-damaging agent and an established human carcinogen. The EF-1H complex also binds to transplatin modified DNA but not to cisplatin-modified DNA. These results demonstrate that the EF-1H complex has functional DNA binding activity and is capable of recognizing the distortions in DNA structure resulting from the covalent binding of Cr and transplatin to DNA.

Amino Acid Sequence↗
PubMed1997

L-arginine potentiates GABA-mediated synaptic transmission by a nitric oxide-independent mechanism in rat dopamine neurons.

Effects of L-arginine in the nervous system are often attributed to nitric oxide. Using whole-cell patch pipettes to record membrane currents in voltage-clamp from dopamine neurons in the rat midbrain slice, the present studies found that L-arginine potentiates GABA-dependent membrane currents via a nitric oxide-independent mechanism. L-Arginine (0.3-10 mM) increased the peak amplitude, half-width duration and time constant of decay of GABA(B) receptor-mediated inhibitory postsynaptic currents in a concentration-dependent manner. In the presence of CGP 35348 (300 microM), a GABA(B) receptor antagonist, L-arginine also prolonged the duration of inhibitory postsynaptic currents mediated by GABA(A) receptors, but their amplitudes were reduced. L-Arginine (10 mM) also evoked 17+/-3 pA of outward current (at -60 mV) which was significantly increased in the presence of exogenous GABA (100 microM). Pressure-ejection of GABA from micropipettes produced outward currents mediated by GABA(B) receptors (recorded in bicuculline) or GABA(A) receptors (recorded in CGP 35348); both types of receptor-mediated currents were increased by L-arginine (10 mM). In contrast, outward currents evoked by baclofen, a GABA(B) receptor agonist, were not potentiated by L-arginine. The GABA transport inhibitors NO 711 (1 microM) and nipecotic acid (1 mM) significantly increased the half-width duration and time-constant of decay of GABA(B)-mediated inhibitory postsynaptic currents, thus mimicking effects of L-arginine. However, nitric oxide donors failed to mimic effects of L-arginine on GABA(B) inhibitory postsynaptic currents, and inhibitors of nitric oxide synthesis failed to selectively block the action of L-arginine. These findings suggest that L-arginine potentiates GABA synaptic transmission by a nitric oxide-independent mechanism. Similarities between effects of L-arginine, NO 711 and nipecotic acid suggest that L-arginine inhibits a GABA transporter.

Animals↗
PubMed1997

A slow excitatory postsynaptic current mediated by G-protein-coupled metabotropic glutamate receptors in rat ventral tegmental dopamine neurons.

Dopamine neurons in the substantia nigra and ventral tegmental area express metabotropic glutamate receptors, but activation of these receptors by synaptic release of neurotransmitter has not been demonstrated thus far. Patch pipettes were used to record membrane currents under voltage clamp from presumed dopamine-containing neurons in the whole-cell configuration in the rat brain slice. A short train of electrical stimuli delivered to bipolar electrodes placed in the slice evoked a slow excitatory postsynaptic current (EPSC; 50-300 pA at -70 mV) which peaked 560 ms after onset and lasted several seconds, with a decay time-constant of 630 ms. This slow EPSC was voltage-dependent, and was abolished by tetrodotoxin (0.5 microM) or by perfusate containing low calcium (0.5 mM) and high magnesium (10 mM). The metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG; 300 microM) blocked the slow EPSC, but L(+)-2-amino-3-phosphonopropionic acid (AP3; 300 microM) had no effect. The slow EPSC was largely occluded by inward current produced by the metabotropic receptor agonist trans-(+/-)-1-amino-1, 3-cyclopentanedicarboxylic acid (t-ACPD; 300 microM), and the EPSC was reduced > 90% during acute desensitization produced by prolonged perfusion with t-ACPD. (+/-)-2-Amino-4-phosphonobutyric acid (AP4; 300 microM), another metabotropic receptor agonist, reduced the slow EPSC but had no effect on currents evoked by t-ACPD applied by pressure-ejection from micropipettes. The slow EPSC was progressively reduced in amplitude when pipettes contained the G-protein inhibitor GDP-beta-S (0.5 mM). When pipettes contained GTP-gamma-S (0.5 mM), a non-hydrolysable analogue of GTP, onset of the slow EPSC was more rapid and its decay was significantly prolonged. These results demonstrate that a slow EPSC mediated by G-protein-coupled metabotropic glutamate receptors can be evoked in dopamine neurons.

Animals↗