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J Hescheler

Publications and source records attributed to J Hescheler.

At least 37 records · Page 2Linked to original sources
PubMed2001

Disruption of cytoskeletal integrity impairs Gi-mediated signaling due to displacement of Gi proteins.

beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.

Acetylcholine↗
PubMed2001

Local response of L-type Ca(2+) current to nitric oxide in frog ventricular myocytes.

1. The regulation of L-type Ca(2+) current (I(Ca)) by the two nitric oxide (NO) donors sodium nitroprusside (SNP, 1 microM to 1 mM) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 3 or 10 microM) was investigated in frog ventricular myocytes using double voltage clamp and double-barrelled microperfusion techniques. 2. SNP and SNAP depressed the isoprenaline (ISO, 10-100 nM)- or forskolin (FSK, 1 microM)-mediated stimulation of I(Ca) via cGMP activation of the cGMP-stimulated phosphodiesterase (PDE2). Complete inhibition of the ISO (100 nM) response was observed at 1 mM SNP. 3. When SNP was applied locally, i.e. to one-half of the cell, and ISO to the whole cell, the response of I(Ca) to ISO was strongly antagonized in the cell half exposed to SNP (up to 100 % inhibition at 1 mM SNP) but a relatively small depression was observed in the other half of the cell (only 20 % inhibition at 1 mM SNP). 4. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO, 1 mM) reversed the local effect of SNAP (3 microM) on FSK-stimulated I(Ca) when applied to the same side as the NO donor, but had no effect when applied to the other side of the cell. 5. A local application of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 30 microM), a selective inhibitor of PDE2, fully reversed the local effect of SNP (100 microM) or SNAP (10 microM) on I(Ca) but had no effect on the distant response. 6. When EHNA was applied on the distant side, with SNP (1 mM) and ISO (100 nM) applied locally, the distant effect of SNP was fully reversed. 7. Our results demonstrate that in frog ventricular myocytes stimulation of guanylyl cyclase by NO leads to a strong local depletion of cAMP near the L-type Ca(2+) channels due to activation of PDE2, but only to a modest reduction of cAMP in the rest of the cell. This may be explained by the existence of a tight microdomain between L-type Ca(2+) channels and PDE2.

Adenine↗
PubMed2001

Down-regulation of intrinsic P-glycoprotein expression in multicellular prostate tumor spheroids by reactive oxygen species.

Intrinsic expression of the multidrug resistance (MDR) transporter P-glycoprotein (Pgp) may be regulated by reactive oxygen species (ROS). A transient expression of Pgp was observed during the growth of multicellular tumor spheroids. Maximum Pgp expression occurred in tumor spheroids with a high percentage of quiescent, Ki-67-negative cells, elevated glutathione levels, increased expression of the cyclin-dependent kinase inhibitors p27Kip1 and p21WAF-1 as well as reduced ROS levels and minor activity of the mitogen-activated kinase (MAPK) members c-Jun amino-terminal kinase (JNK), extracellular signal-regulated kinase ERK1,2, and p38 MAPK. Raising intracellular ROS by depletion of glutathione with buthionine sulfoximine (BSO) or glutamine starvation resulted in down-regulation of Pgp and p27Kip1, whereas ERK1,2 and JNK were activated. Down-regulation of Pgp was furthermore observed with low concentrations of hydrogen peroxide and epidermal growth factor, indicating that ROS may regulate Pgp expression. The down-regulation of Pgp following BSO treatment was abolished by agents interfering with receptor tyrosine kinase signaling pathways, i.e. the protein kinase C inhibitors bisindolylmaleimide I (BIM-1) and Ro-31-8220, the p21ras farnesyl protein transferase inhibitor III, the c-Raf inhibitor ZM 336372 and PD98059, which inhibits ERK1,2 activation. ROS involved as second messengers in receptor tyrosine kinase signaling pathways may act as negative regulators of Pgp expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Protein and mRNA expression of serum and glucocorticoid-dependent kinase 1 in metanephrogenesis.

Abstract Expression of serum and glucocorticoid-dependent kinase 1 (SGK1) during development in mouse kidney (embryonic day [E] 14 to postnatal day [P] 1) was studied by in situ hybridization and immunofluorescence. In whole embryos, SGK1 mRNA was highly abundant in the developing metanephros, where SGK1 mRNA was expressed in the ureteric buds of the branching collecting duct system and in the mesenchymal blastema-derived comma- and s-shaped bodies. In E14 kidneys, SGK1 protein was below detection level, whereas at day E16, ureteric buds, s-shaped bodies and outgrowing loops of Henle expressed detectable amounts of SGK1 protein. SGK1 protein was also expressed in E16 primary tubules of the collecting duct system. In P1 kidneys, no or only faint SGK1 protein expression was apparent in comma- and s-shaped bodies, whereas SGK1 was continuously expressed by medullary collecting ducts. In conclusion, SGK1 is developmentally expressed in metanephrogenesis. High expression in developing collecting duct and in blastema-derived comma- and s-shaped bodies suggests a dual function of SGK1 in maturation of the reabsorbing collecting duct epithelium and in epithelial transition of the blastema cells.

Animals↗
PubMed2001

The expression and regulation of depolarization-activated K+ channels in the insulin-secreting cell line INS-1.

The aim of the present study was to characterize depolarization-activated outward currents in insulin-secreting INS-1 cells and to investigate the role of K+ channels other than the KATP channels in the regulation of insulin release. Outward currents were inhibited by 4-aminopyridine (4-AP, 10 mmol/l), tetraethylammonium (TEA, 10 mmol/l) and tetrapentylammonium (TPeA, 100 mumol/l) by 55.1 +/- 3.8% (n = 3), 78.1 +/- 3.2% (n = 6) and 98.7 +/- 0.8% (n = 5), respectively. Margatoxin (5 nmol/l) and charybdotoxin (3 mumol/l) had no effect. 4-AP inhibited mainly a fast-activating, slowly inactivating current, whereas the TEA- and TPeA-sensitive current components were slowly activating and non-inactivating. Forskolin and the forskolin analogue 1,9-dideoxyforskolin, which does not stimulate adenylyl cyclase, also inhibited the outward current, suggesting a direct effect on the channels. Using reverse transcriptase polymerase chain reaction (RT/PCR). Kv channel mRNAs of Kv1.4, Kv1.5, Kv2.1, Kv2.2, Kv3.1 and Kv3.2 were detected whereas other Kv channels, Kv1.1, Kv1.2, Kv1.3, Kv1.6 and Kv3.4 were not detected. Insulin secretion in the presence of tolbutamide (100 mumol/l) was increased by 4-AP, TEA and TPeA by 65%, 41% and 150%, respectively. Basal secretion was not affected by these blockers. Our study reveals that the opening of voltage-dependent K+ channels negatively controls insulin secretion in depolarized cells, probably by shortening the action potential thus reducing Ca2+ influx.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Establishment of an in vitro reporter gene assay for developmental cardiac toxicity.

This study is based on the unique potential of pluripotent embryonic stem (ES) cells to differentiate in vitro into embryoid bodies containing cell lineages representative of most cell types found in the mammalian fetus. However, the use of wild type ES cells as an in vitro assay for embryotoxicological studies is complicated by the simultaneous development of various cellular phenotypes. This prevents a quantitative assessment of drug effects on one specific cell type. Here we report the effects of 15 chemicals on cardiac differentiation as determined by various specific toxicological endpoints such as morphological inspection (contractile activity), quantitative mRNA analysis and cardiac-specific expression of green fluorescent protein (GFP), used as a quantitative reporter. The data from the different endpoints have been subjected to a statistical analysis, and a preliminary prediction model is proposed. The results demonstrate that genetically-engineered ES cells could provide a valuable tool for estimating the developmental cardiotoxic potential of compounds in vitro and form the basis for automated analysis in a high-throughput system.

Animals↗
PubMed2001

Functional coupling between 'R-type' Ca2+ channels and insulin secretion in the insulinoma cell line INS-1.

Among voltage-gated Ca2+ channels the non-dihydropyridine-sensitive alpha1E subunit is functionally less well characterized than the structurally related alpha1A (omega-agatoxin-IVA sensitive, P- /Q-type) and alpha1B (omega-conotoxin-GVIA sensitive, N-type) subunits. In the rat insulinoma cell line, INS-1, a tissue-specific splice variant of alpha1E (alpha1Ee) has been characterized at the mRNA and protein levels, suggesting that INS-1 cells are a suitable model for investigating the function of alpha1Ee. In alpha1E-transfected human embryonic kidney (HEK-293) cells the alpha1E-selective peptide antagonist SNX-482 (100 nM) reduces alpha1Ed- and alpha1Ee-induced Ba2+ inward currents in the absence and presence of the auxiliary subunits beta3 and alpha2delta-2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX-482 was detected on the recombinant T-type Ca2+ channel subunits alpha1G, alpha1H, and alpha1I showing that the toxin from the venom of Hysterocrates gigas is useful as an alpha1E-selective antagonist. After blocking known components of Ca2+ channel inward current in INS-1 cells by 2 microM (+/-)-isradipine plus 0.5 microM omega-conotoxin-MVIIC, the remaining current is reduced by 100 nM SNX-482 from -12.4 +/- 1.2 pA/pF to -7.6 +/- 0.5 pA/pF (n = 9). Furthermore, in INS-1 cells, glucose- and KCl-induced insulin release are reduced by SNX-482 in a dose-dependent manner leading to the conclusion that alpha1E, in addition to L-type and non-L-type (alpha1A-mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS-1 cells.

Alternative Splicing↗
PubMed2001

Tumor-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells.

Tumor vascularization is the rate-limiting step for the progression of cancer. Differential steps of tumor-induced angiogenesis were studied by a novel in vitro confrontation culture of avascular multicellular prostate tumor spheroids and embryoid bodies grown from pluripotent embryonic stem (ES) cells. Vascularization in embryoid bodies started on day 5 of cell culture and was paralleled by down-regulation of hypoxia-inducible factor 1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF). In parallel, a dissipation of gradients in the pericellular oxygen pressure was observed as measured by O(2)-sensitive microelectrodes. After 24--48 h of confrontation culture, cells positive for platelet endothelial cell adhesion molecule (PECAM-1) became visible in the contact region between the embryoid body and the tumor spheroid and sprouted within the confrontation cultures during subsequent days. Tumor-induced angiogenesis resulted in growth stimulation of tumor spheroids, disappearance of central necrosis and a reduction of the pericellular oxygen pressure. Furthermore, tumor vascularization resulted in elevated levels of HIF-1 alpha, VEGF, heat shock protein 27 (HSP27), and P-glycoprotein. Tumor-induced angiogenesis may augment the oxygen consumption in tumors resulting in an increased expression of hypoxia-related, proangiogenic genes as well as of HSP27 and P-glycoprotein, which are involved in a multidrug resistance phenotype.

Animals↗
PubMed2001

Loss of annexin A7 leads to alterations in frequency-induced shortening of isolated murine cardiomyocytes.

Annexin A7 has been proposed to function in the fusion of vesicles, acting as a Ca(2+) channel and as Ca(2+)-activated GTPase, thus inducing Ca(2+)/GTP-dependent secretory events. To understand the function of annexin A7, we have performed targeted disruption of the Anxa7 gene in mice. Matings between heterozygous mice produced offspring showing a normal Mendelian pattern of inheritance, indicating that the loss of annexin A7 did not interfere with viability in utero. Mice lacking annexin A7 showed no obvious phenotype and were fertile. To assay for exocytosis, insulin secretion from isolated islets of Langerhans was examined. Ca(2+)-induced and cyclic AMP-mediated potentiation of insulin secretion was unchanged in the absence of annexin A7, suggesting that it is not directly implicated in vesicle fusion. Ca(2+) regulation studied in isolated cardiomyocytes, showed that while cells from early embryos displayed intact Ca(2+) homeostasis and expressed all of the components required for excitation-contraction coupling, cardiomyocytes from adult Anxa7(-/-) mice exhibited an altered cell shortening-frequency relationship when stimulated with high frequencies. This suggests a function for annexin A7 in electromechanical coupling, probably through Ca(2+) homoeostasis.

Animals↗
PubMed2001

Characteristics of calcium sparks in cardiomyocytes derived from embryonic stem cells.

In embryonic stem (ES) cell-derived cardiomyocytes, spontaneous Ca(2+) sparks representing Ca(2+) release through ryanodine receptor (RyR) channels were characterized and correlated to the expression of RyRs as well as the Ca(2+) load of the sarcoplasmic reticulum (SR). In very early developmental stage (VEDS) cardiac precursor cells, global intracellular Ca(2+) concentration ([Ca(2+)](i)) fluctuations occurred, whereas Ca(2+) sparks and contractions were absent. In early developmental stages (EDS), contractions as well as Ca(2+) sparks were obvious. During the further differentiation to late developmental stage (LDS) cardiomyocytes, a marked increase in the frequency of global [Ca(2+)](i) transients, the amplitude and the frequency of Ca(2+) sparks, as well as the expression of RyRs and the volume of RyR-positive SR, was observed. Furthermore, the caffeine-releasable SR Ca(2+) load was elevated in LDS compared with EDS cardiomyocytes. A high-Ca(2+) solution raised spark frequency as well as amplitude in EDS cardiomyocytes to the levels of LDS cardiomyocytes. The characteristics of Ca(2+) sparks occurring in cardiomyocytes differentiated from ES cells may be governed by the Ca(2+) load of the SR and/or the density of RyRs.

Animals↗
PubMed2001

Reactive oxygen species as intracellular messengers during cell growth and differentiation.

Reactive oxygen species (ROS) are generated following ligand-receptor interactions and function as specific second messengers in signaling cascades involved in cell proliferation and differentiation. Although ROS are generated intracellularly by several sources, including mitochondria, the primary sources of ROS involved in receptor-mediated signaling cascades are plasma membrane oxidases, preferentially NADPH oxidases, with a rapid kinetics of activation and inactivation. This allows a tight up- and downregulation of intracellular ROS levels within the short time required for the transduction of signals from the plasma membrane to the cell nucleus. The mode of action of ROS may involve direct interaction with specific receptors, and/or redox-activation of members of signaling pathways such as protein kinases, protein phosphatases, and transcription factors. Furthermore, ROS act in concert with intracellular Ca(2+) in signaling pathways which regulate the balance of cell proliferation versus cell cycle arrest and cell death. The delicate intracellular interplay between oxidizing and reducing equivalents allows ROS to function as second messengers in the control of cell proliferation and differentiation.

Animals↗
PubMed2001

Gangliosides GM1 and GM2 induce vascular smooth muscle cell proliferation via extracellular signal-regulated kinase 1/2 pathway.

Gangliosides, sialic acid-containing glycophospholipids, accumulate in atherosclerotic vessels and appear to regulate the proliferation of various cell types. Furthermore, vascular smooth muscle cell (VSMC) proliferation is associated with the development and progression of cardiovascular diseases. To demonstrate whether gangliosides are able to modulate the VSMC growth, the effect of gangliosides GM1, GM2, and GM3 on cell DNA synthesis and cell number has been examined. Moreover, we investigated possible intracellular mechanisms by which GM1 and GM2 elicit their mitogenic effects. Stimulation of VSMCs with GM1 and GM2 resulted in a dose-dependent increase in DNA synthesis and cell number, whereas GM3 caused a decrease in DNA synthesis. GM1 and GM2 (50 micromol/L) stimulate phosphorylation of extracellular signal-regulated kinases (ERKs) 1 and 2 and phosphorylation of the c-Jun N-terminal kinase (JNK), with a maximum at 15 minutes, but they do not have an effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK). GM3 (50 micromol/L), on the other hand, does not stimulate any of the 3 aforementioned MAPKs. Pretreatment of the cells with 20 micromol/L PD 098,059 caused a complete inhibition of ERK1/2 and JNK MAPK, whereas pretreatment with a Ras (farnesyl transferase) inhibitor did not abrogate the GM1- and GM2-induced ERK1/2 phosphorylation. Furthermore, GM1 and GM2 did not activate Raf-1 kinase. Interestingly, pretreatment of VSMCs with 100 nmol/L pertussis toxin resulted in a complete inhibition of the ERK1/2 phosphorylation. Finally, the GM1- and GM2-induced increase in cell number was significantly inhibited by PD 098,059. We may conclude that GM1 and GM2 stimulate ERK1/2 via a pertussis toxin-sensitive G(i)-coupled receptor through a Raf-1 kinase-independent pathway. Moreover, the GM1- and GM2-induced VSMC growth is ERK1/2 dependent.

Animals↗
PubMed2001

Measurement of apoptosis in long-term cultures of human ovarian tissue.

At present, the long-term culture of ovarian tissue is problematic. The aim of this study was to measure apoptosis in long-term cultures of human ovarian tissue. Biopsies of human ovaries were cultured for 6 weeks. Samples were taken weekly for histological investigation. The apoptotic cells were marked with anti-caspase 3. Simultaneous to this experiment, other tissue samples were preincubated for 3 h with 1 micromol staurosporine l(-1), an inducer of apoptosis, and apoptosis was compared among samples. Furthermore, the proportion of lethal cells was determined weekly. After 6 weeks, 99% of the tissue samples showed an intact structure. They expanded in all directions on the floor of the multi-wells to form a monolayer. Apoptotic cells could be marked only sporadically (16.3 +/- 5.9 fluorescence (counts per 3600 microm(2))) after 6 weeks. After preincubation with staurosporine after the same period of culture, the proportion of apoptotic cells was significantly increased compared with that in untreated control samples (66.8 +/- 14.5 versus 16.3 +/- 5.9%, respectively; P < 0.05). Under the same experimental conditions, the proportion of lethal cells was 3.6 +/- 0.9, 3.9 +/- 2.1 and 5.2 +/- 1.5% for weeks 1, 3 and 6, respectively. After preincubation with 1 micromol staurosporine l(-1), the proportion of pyknotic cells after 6 weeks of culture was significantly higher (37.2 +/- 4.4%) than that in control samples (3.95 +/- 2.05%; P < 0.05). No significant increase in apoptosis in cultured human ovarian tissue after 6 weeks was observed compared with control tissues on day 1. These results indicate that under optimal culture conditions it is possible to cultivate human ovarian tissue long term. The influence of long-term culture on hormone synthesis and follicle maturity will be investigated further.

Adult↗
PubMed2001

Nestin-specific green fluorescent protein expression in embryonic stem cell-derived neural precursor cells used for transplantation.

Expression of the enhanced green fluorescent protein (EGFP) under control of a thymidine kinase promoter/nestin second intron was specifically detected in nestin immunoreactive neural precursor cells after selection of murine embryonic stem (ES) cells in chemically defined medium. Allowing differentiation in vitro, the capacity of these cells to give rise to astroglia, oligodendroglia, and neurones was investigated. After intracerebral transplantation, long-lasting integration of precursor cells into the host tissue was observed, serving as a pool for successive neuronal and glial differentiation. EGFP expression by ES cell-derived neural precursor cells may be a valuable tool to optimize protocols for maintenance and expansion of these cells in vitro as well as in vivo after intracerebral transplantation. In addition, preparative fluorescence-activated cell sorting of EGFP-labeled neural precursor cells should be useful for standardization of a donor cell population for cell replacement therapies.

Animals↗
PubMed2000

Role of reactive oxygen species and phosphatidylinositol 3-kinase in cardiomyocyte differentiation of embryonic stem cells.

Cardiotypic development in embryonic stem cell-derived embryoid bodies may be regulated by reactive oxygen species (ROS). ROS were generated by a NADPH oxidase-like enzyme which was transiently expressed during the time course of embryoid body development. Incubation with either H(2)O(2) or menadione enhanced cardiomyogenesis, whereas the radical scavengers trolox, pyrrolidinedithiocarbamate and N-acetylcysteine exerted inhibitory effects. The phosphatidylinositol 3-kinase (PI-3-kinase) inhibitors LY294002 and wortmannin abolished cardiac commitment and downregulated ROS in embryoid bodies. Coadministration of LY294002 with prooxidants resumed cardiomyocyte differentiation, indicating a role for PI-3-kinase in the regulation of the intracellular redox state.

Androstadienes↗
PubMed2000

Functional expression and regulation of the hyperpolarization activated non-selective cation current in embryonic stem cell-derived cardiomyocytes.

1. The biophysical and pharmacological characteristics of the hyperpolarization activated non- selective cation current (If) were recorded using whole-cell voltage clamp in embryonic stem (ES) cell-derived cardiomyocytes at different stages of development. 2. The cation current was detected in a large percentage (65 %) of early stage (EDS, differentiated for 7 + 3-4 days) cells at a current density of 11.4 +/- 0.6 pA pF-1 (n = 47). In late stage (LDS, differentiated for 7 + 9-12 days) cells the percentage of cells expressing If decreased (45 %), but If densities (15.5 +/- 0.9 pA pF-1, n = 20) were increased. 3. The muscarinic agonist carbachol (CCh, 1 microM) depressed basal If in EDS cells by 45.7 +/- 6.5 %, n = 5) and was without effect in LDS cardiomyocytes (n = 4). The beta-adrenoceptor agonist isoprenaline (ISO, 1 microM) stimulated If in LDS cells by 33 +/- 5.2 % (n = 6) but not in EDS cells (n = 5). 4. Cell infusion with the catalytic subunit of the cAMP-dependent protein kinase (PKA, 7 microM) stimulated If in EDS cells by 37.0 +/- 2.9 %, (n = 4), but subsequent superfusion of 8-bromo-cAMP (200 microM) was without effect. Intracellular perfusion of LDS cardiomyocytes with the highly selective peptide inhibitor of PKA (PKI, 20 microM) completely inhibited the stimulation of the L-type Ca2+ current (ICa,L) as well as of If by ISO (1 microM). 5. Extracellular superfusion with phosphodiesterase (PDE) inhibitors - IBMX, a non-selective antagonist, Erythro-9-(2-hydoxy-3-nonyl)adenine (EHNA), a PDE2 antagonist and rolipram, a PDE4 antagonist - resulted in stimulation of ICa,L and If in EDS cells. By contrast, milrinone and cilostamide, two PDE3 antagonists, stimulated ICa,L, but not If. 6. The present work demonstrates that If is functionally expressed during early cardiomyogenesis. Similar to ICa,L, If is regulated during embryonic development by phosphorylation via PKA. In contrast to ICa,L, If is not regulated by PDE3 suggesting different localization of these ion channels with respect to PDE3.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Redox regulation of P-glycoprotein-mediated multidrug resistance in multicellular prostate tumor spheroids.

Multicellular prostate tumor spheroids develop intrinsic P-glycoprotein (Pgp)-mediated multidrug resistance with the appearance of quiescent cell areas. We have investigated the effect of intracellular reactive oxygen species (ROS) on Pgp expression in large, quiescent and drug-resistant multicellular spheroids (diameter 250 +/- 50microm). Using the ROS-sensitive fluorescence dye 2;7;-dichlorodihydrofluorescein diacetate (H(2)DCFDA), we demonstrated that these tumor spheroids are characterized by reduced intracellular ROS compared with drug-sensitive small spheroids (diameter 60 +/- 20microm) consisting predominantly of proliferating cells. The prooxidants hydrogen peroxide, menadione and glyceraldehyde raised ROS in large tumor spheroids and significantly down-regulated Pgp within 24 hr. Comparable effects were achieved with the known Pgp-reversing agents sodium orthovanadate, quinidine and cyclosporin A but not with verapamil. Consequently, the retention and toxicity of the anthracycline doxorubicin was increased in tumor spheroids treated with prooxidants. Co-administration of prooxidants and the free radical scavenger ebselen did not alter Pgp levels, indicating that down-regulation of Pgp is mediated via ROS. Down-regulation of Pgp by H(2)O(2) was abolished when either forskolin, 8-Br-cAMP or IBMX, which raise intracellular cAMP levels, was co-administered, indicating that Pgp expression is regulated by protein kinase A (PKA). Furthermore, Pgp was down-regulated by the PKA inhibitors Rp-cAMPs and H89. Since prooxidants stimulated the growth of multicellular spheroids and down-regulated the cyclin-dependent kinase inhibitor p27(kip1), we conclude that ROS-mediated Pgp down-regulation may be paralleled by recruitment of drug-resistant quiescent cells in the depth of the tumor tissue for cell-cycle activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗