Human multidrug-resistant cell lines: increased mdr1 expression can precede gene amplification.

The development of simultaneous resistance to multiple structurally unrelated drugs is a major impediment to cancer chemotherapy. Multidrug resistance in human KB carcinoma cells selected in colchicine, vinblastine, or Adriamycin is associated with amplification of specific DNA sequences (the multidrug resistance locus, mdr1). During colchicine selection resistance is initially accompanied by elevated expression of a 4.5-kilobase mdr1 messenger RNA (mRNA) without amplification of the corresponding genomic sequences. During selection for increased levels of resistance, expression of this mRNA is increased simultaneously with amplification of mdr1 DNA. Increased expression and amplification of mdr1 sequences were also found in multidrug-resistant sublines of human leukemia and ovarian carcinoma cells. These results suggest that increased expression of mdr1 mRNA is a common mechanism for multidrug resistance in human cells. Activation of the mdr1 gene by mutations or epigenetic changes may precede its amplification during the development of resistance.

Structure and expression of the human MDR (P-glycoprotein) gene family.

The human MDR (P-glycoprotein) gene family is known to include two members, MDR1 and MDR2. The product of the MDR1 gene, which is responsible for resistance to different cytotoxic drugs (multidrug resistance), appears to serve as an energy-dependent efflux pump for various lipophilic compounds. The function of the MDR2 gene remains unknown. We have examined the structure of the human MDR gene family by Southern hybridization of DNA from different multidrug-resistant cell lines with subfragments of MDR1 cDNA and by cloning and sequencing of genomic fragments. We have found no evidence for any other cross-hybridizing MDR genes. The sequence of two exons of the MDR2 gene was determined from genomic clones. Hybridization with single-exon probes showed that the human MDR1 gene is closely related to two genes in mouse and hamster DNA, whereas MDR2 corresponds to one rodent gene. The human MDR locus was mapped by field-inversion gel electrophoresis, and both MDR genes were found to be linked within 330 kilobases. The expression patterns of the human MDR genes were examined by enzymatic amplification of cDNA. In multidrug-resistant cell lines, increased expression of MDR1 mRNA was paralleled by a smaller increase in the levels of MDR2 mRNA. In normal human tissues, MDR2 was coexpressed with MDR1 in the liver, kidney, adrenal gland, and spleen. MDR1 expression was also detected in colon, lung, stomach, esophagus, muscle, breast, and bladder.

Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene.

Mouse NIH 3T3 cells were transformed to multidrug resistance with high-molecular-weight DNA from multidrug-resistant human KB carcinoma cells. The patterns of cross resistance to colchicine, vinblastine, and doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.) of the human donor cell line and mouse recipients were similar. The multidrug-resistant human donor cell line contains amplified sequences of the mdr1 gene which are expressed at high levels. Both primary and secondary NIH 3T3 transformants contained and expressed these amplified human mdr1 sequences. Amplification and expression of the human mdr1 sequences and amplification of cotransferred human Alu sequences in the mouse cells correlated with the degree of multidrug resistance. These data suggest that the mdr1 gene is likely to be responsible for multidrug resistance in cultured cells.

Effects of skyrin, a receptor-selective glucagon antagonist, in rat and human hepatocytes.

Peptidic glucagon antagonists have been shown to lower blood glucose levels in diabetic models (1-3), but attempts to identify small molecular weight glucagon receptor-binding antagonists have met with little success. Skyrin, a fungal bisanthroquinone, exhibits functional glucagon antagonism by uncoupling the glucagon receptor from adenylate cyclase activation in rat liver membranes (1). We have examined the effects of skyrin on cells transfected with the human glucagon receptor and on isolated rat and human hepatocytes. The skyrin used was isolated from Talaromyces wortmanni American Type Culture Collection 10517. In rat hepatocytes, skyrin (30 micromol/l) inhibited glucagon-stimulated cAMP production (53%) and glucose output (IC50 56 micromol/l). There was no detectable effect on epinephrine or glucagon-like peptide 1 (GLP-1) stimulation of these parameters, which demonstrates skyrin's selective activity. Skyrin was also evaluated in primary cultures of human hepatocytes. Unlike cell lines, which are largely unresponsive to glucagon, primary human hepatocytes exhibited glucagon-dependent cAMP production for 14 days in culture (EC50 10 nmol/l). Skyrin (10 micromol/l) markedly reduced glucagon-stimulated cAMP production (55%) and glycogenolysis (27%) in human hepatocytes. The inhibition of glucagon stimulation was a specific property displayed by skyrin and oxyskyrin but not shared by other bisanthroquinones. Skyrin is the first small molecular weight nonpeptidic agent demonstrated to interfere with the coupling of glucagon to adenylate cyclase independent of binding to the glucagon receptor. The data presented in this study indicate that functional uncoupling of the human glucagon receptor from cAMP production results in metabolic effects that could reduce hepatocyte glucose production and hence alleviate diabetic hyperglycemia.

Activation of protein kinase C alpha inhibits insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1.

Chinese hamster ovary (CHO) cells were transfected with a cDNA encoding protein kinase C alpha (PKC) and a cell line (CHO-PKC alpha) expressing approximately 7-fold greater amounts of PKC as the parental cells were isolated. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate in the CHO-PKC alpha cells inhibited by approximately 75% the: 1) insulin-stimulated increase in antiphosphotyrosine precipitable phosphatidylinositol 3-kinase activity in these cells; 2) insulin-stimulated increase in PI 3-kinase activity associated with insulin receptor substrate-1; and 3) tyrosine phosphorylation of the endogenous substrate, insulin receptor substrate-1. In contrast, 12-O-tetradecanoylphorbol-13-acetate treatment did not inhibit any of these responses in the parental CHO cells. These results indicate that excessive PKC activity can interfere in a very early step in insulin receptor signaling and are consistent with the hypothesis that excessive PKC activity may contribute to some states of insulin resistance.

Pulmonary ferritin: differential effects of hyperoxic lung injury on subunit mRNA levels.

Ferritin is an iron storage protein that is regulated at the transcriptional and transcriptional levels, resulting in a complex mixture of tissue- and condition-specific isoforms. The protein shell of ferritin is composed of 24 subunits of two types (heavy or light), which are encoded by two distinct and independently regulated genes. In the present studies, the isoform profile for lung ferritin differed from other tissues (liver, spleen, and heart) as determined by isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). Lung ferritin was composed of equal amounts of heavy and light subunits. Differences in isoform profiles were the result of tissue-specific differential expression of the ferritin subunit genes as demonstrated by Northern blot analyses. Like heart ferritin, lung ferritin exhibited a low iron content that did not increase extensively in response to iron challenge, which contrasts with ferritins isolated from liver or spleen. When animals were exposed to hyperoxic conditions (95% oxygen for up to 60 h), ferritin heavy subunit mRNA levels did not markedly change at any of the investigated time points. In contrast, ferritin light subunit mRNA increased severalfold in response to hyperoxic exposure. Investigation of the cytoplasmic distribution of ferritin mRNA showed that a substantial portion was associated with the ribonucleoprotein (RNP) fraction of the cytosol, suggesting that a pool of untranslated ferritin mRNA exists in the lung. Upon hyperoxic insult, all ferritin light subunit mRNA pools (RNP, monosomal, polysomal) were elevated, although a specific shift from RNP to polysomal pools was not evident. Therefore, the increase in translatable ferritin mRNA in response to hyperoxia resulted from transcriptional rather than specific translational activation. The observed pattern of light chain-specific transcriptional induction of ferritin is consistent with the hypothesis that hyperoxic lung injury is at least partially iron mediated.

Flow cytometric detection of antigen-specific cytokine responses in lung T cells in a murine model of pulmonary inflammation.

Multiparameter flow cytometry was used to examine the cytokine responses of antigen-specific T lymphocytes isolated from the lungs of antigen-sensitized mice which developed pulmonary inflammation after aerosol challenge with ovalbumin (OA) (OA/OA). Lung T cells were stimulated in vitro with OA and anti-CD28 monoclonal antibody (mAb) in the presence of the secretion inhibitor, brefeldin A. T cell subsets were examined for intracellular cytokine expression using fluorochrome-labeled cell-surface specific and anti-cytokine antibodies. Antigen-specific responses resulted in significant numbers of CD4+ lung cells expressing cytoplasmic interleukin (IL)-2 (6%), IL-4 (1.5%), IL-5 (4%), and tumor necrosis factor (TNF)-alpha (11%), but not interferon (IFN)-gamma. Dual cytokine analyses demonstrated antigen-specific responses resulted in CD4+ T cells being positive for IL-2 and IL-4 or IL-2 and IL-5. TNF-alpha was the only antigen-specific cytokine response detected in CD8+ lung T cells after in vitro activation with OA. Cytokines in the supernatants of cultures activated with OA and anti-CD28 were measured by ELISA and the results confirmed the antigen-specific responses measured by flow cytometry. Polyclonal activation of lung T cells from OA/OA mice with 12-myristate 13-acetate (PMA), ionomycin, anti-CD3 mAb, and anti-CD28 mAb resulted in higher percentages of IL-2+ (43%) and IL-5+ (7%) CD4 cells when compared to CD4+ T cells from non-OA sensitized, challenged mice. CD8+ cells from OA/OA mice demonstrated intracellular staining for IL-2 (26%), TNF-alpha (55%), and IFN-gamma (37%), but not IL-4 or IL-5, after polyclonal activation. There is less agreement between intracellular cytokine staining of CD4+ T cells and cytokines released into the culture medium after polyclonal activation. Dual cytokine analyses of polyclonal-activated CD4+ cells demonstrated co-expression of IFN-gamma with IL-2, IL-4, or IL-5. T cells co-expressing IL-2 with IL-4 or IL-5 were also detected. These results demonstrate the utility of multiparameter flow cytometry to directly measure antigen-specific cytokine responses in subsets of T lymphocytes isolated from inflammatory sites.

Attenuation of oxidant-induced lung injury by 21-aminosteroids (lazaroids): correlation with the mRNA expression for E-selectin, P-selectin, ICAM-1, and VCAM-1.

We compared the effects of treatment with methylprednisolone or the 21-aminosteroids, U-74389 and U-74006F (Tirilizad mesylate), on hyperoxic lung injury and the associated expression of mRNA for several adhesion molecules in rats. Inhalation of > 95% oxygen for up to 72 hr in Sprague-Dawley rats produced a marked increase in lung weight and an accumulation of fluid in the thorax when compared with air-breathing controls. Hyperoxia also induced a marked neutrophil-rich influx of inflammatory cells into the bronchial lumen as measured by bronchoalveolar lavage. Neutrophil numbers in bronchoalveolar lavage fluid peaked after 60 hr of exposure to s 95% oxygen; this was associated with a marked upregulation of mRNA for the adhesion molecules P-selectin and E-selectin but not VCAM-1. mRNA for ICAM-1 was constitutively expressed at high levels in both air-breathing controls and in the lungs of rats exposed to high concentrations of oxygen. Pretreatment with the 21-aminosteroids reduced hyperoxic lung damage and improved survival times in animals exposed to > 95% oxygen. However, treatment with methylprednisolone significantly decreased survival times. Treatment with U-74389 did not significantly (p > 0.05) inhibit the BAL neutrophilia and did not significantly (p > 0.05) reduce hyperoxia-induced increases in mRNA expression for P-selectin and E-selectin. The inhibition of hyperoxic lung damage coupled with improved survival seen in treated animals suggests that 21-aminosteroids may provide valuable treatments for pulmonary disorders in which oxidant damage has been implicated.

Endogenous brain cytokine mRNA and inflammatory responses to lipopolysaccharide are elevated in the Tg2576 transgenic mouse model of Alzheimer's disease.

Beta-amyloid (Abeta) plaques have been shown to induce inflammatory changes in Alzheimer's disease brains. Cortical, but not cerebellar tissue from 16-month-old Tg2576 (Tg+) mice showed significant increases in interleukin (IL)-1alpha (2.2-fold), IL-1beta (3.4-fold), tumor necrosis factor-alpha (3.9-fold), and monocyte chemoattractant protein-1 (2.5-fold) mRNA levels compared to controls (Tg-). These changes were not apparent in 6-month-old Tg+ mice except for TNF-alpha. mRNA levels of glial fibrillary acidic protein and complement components, C1qA and C3 were also elevated in aged mice. Lipopolysaccharide (LPS) (25 microg/mouse, i.v.) induced a significantly greater production of IL-1beta protein in the cortices and hippocampi of Tg+ vs. Tg- mice at 1, 2, 4, and 6 h. Experiments in 6-month-old mice showed that not only was there less cytokine produced compared to 16-month-old mice, but the exacerbated cytokine response to LPS in Tg+ mice was not apparent. Higher levels of Abeta1-40 were measured in the cortices of 6- and 16-month-old Tg+ mice at 4-6 h after LPS, which returned to baseline after 18 h. We demonstrate that Abeta plaques elicit inflammatory responses in Tg2576 mice that are further exacerbated when challenged by an exogenous inflammatory insult, which may serve to amplify degenerative processes.

Pattern of cytokine and adhesion molecule mRNA in hapten-induced relapsing colon inflammation in the rat.

We examined the mRNA expression of cytokines, chemokines, integrins, and selectins in colon lesions of rat colitis with a semi-quantitative RT-PCR assay. Rat colitis was induced by trinitrobenzene sulfonic acid (TNBS) in 50% ethanol. Within 24 h, an acute inflammation occurred with hyperemia, edema, necrosis and an intense infiltration of granulocytes in the mucosa. The lesion proceeded into a T-lymphocyte/monocyte-driven chronic inflammation for two weeks and healed in 6 weeks. An acute inflammation recurred at the same site when the recovered animals were systemically injected with TNBS. We isolated RNA from colon tissue at 24 h, 1, 2, 4, 6 weeks after TNBS treatment and from the relapsed animals. The mRNA for cytokines IL-1beta, IL-6, IL-10 and the chemokines CINC, MIP-1alpha, MCP-1 were significantly (P < 0.05) elevated and persisted for 2 weeks, decreased in 6 weeks and increased again during relapse. IFN-gamma mRNA stayed at control levels initially, but increased dramatically in the second weeks of chronic inflammation as well as in relapse. The mRNA levels of adhesion molecules, ICAM-1, VCAM-1, the mucosal homing integrin beta7 as well as P- and E-selectin were greatly enhanced between 1 and 3 weeks. The data showed that the chronically inflamed tissue expresses a time-dependent changing pattern of TH1 cytokines and adhesion molecules that maintain the infiltration and activation of inflammatory cells and tissue injury.

Effects of recombinant human interleukin-1 beta on rabbit articular chondrocytes. Stimulation of prostanoid release and inhibition of cell growth.

Using an in vitro model, we characterized the production of prostaglandin E2, 6-keto-prostaglandin F1 alpha, and thromboxane B2 by normal rabbit articular chondrocytes stimulated by interleukin-1 beta. The prostanoids were produced in a dose- and time-dependent manner, which was sensitive to actinomycin D and cycloheximide. Interleukin-1 also had a pronounced, but reversible, cytostatic effect on chondrocytes, which was not attributable to prostanoid or polyamine synthesis.

Interleukin 1 receptors on rabbit articular chondrocytes: relationship between biological activity and receptor binding kinetics.

This study gives an account of the biologic and kinetic binding properties of interleukin 1 alpha (IL 1 alpha), interleukin 1 beta (IL 1 beta), and Glu-4 (an NH2-terminal mutant of IL 1 beta) to interleukin 1 (IL 1) receptors in rabbit articular chondrocytes. All three IL 1's demonstrated full agonist properties in their ability to stimulate prostaglandin E2 (PGE2) synthesis. IL 1 alpha was 23-fold more biologically active than IL 1 beta, which was around 110-fold more active than Glu-4 based on the concentration of IL 1 required for half-maximal stimulation of PGE2. The binding of all three ligands was concentration-dependent and saturable at 4 degrees C. Scatchard analysis of receptor binding data showed that the dissociation constant (KD) of IL 1 alpha was 46 +/- 12 pM, and the receptor density was 3120 sites/cell. The association of IL 1 alpha at 4 degrees C did not attain equilibrium until after 10 h at 100 pM of 125I-labeled IL 1 alpha. The dissociation of bound IL 1 alpha was very slow, t1/2 of 21 h, although only one class of high-affinity receptors was detected. The KD of IL 1 beta binding was 72 +/- 3 pM with a receptor density of 800 +/- 40 sites/cell. Dissociation of bound 125I-labeled IL 1 beta at 4 degrees C appeared to indicate the presence of two receptor subsets, a fast and a slower component with a t1/2 of 2 min and 5 h, respectively. The receptor binding affinity of Glu-4 was 324 +/- 3 pM, in line with its reduced biologic activity. Both IL 1 alpha and IL 1 beta are rapidly internalized in chondrocytes in a time- and temperature-dependent manner.

Cytokine networks in allergic lung inflammation: an opportunity for drug intervention.

Eosinophils and mast cells have long been considered as the major effector cells ultimately responsible for bronchial obstruction and airway hyper-responsiveness in asthmatics. However, there is now accumulating evidence that products of Th2 lymphocytes may orchestrate the generation, accumulation, and activation of these cells within the airway wall. Since the first report by Mosmannet al. in 1986 that murine helper T-cell clones could be divided into two subsets, Th1 and Th2, depending on their pattern of cytokine secretion, and observations that polarisation of Th1- or Th2-dependent cytokine production could be correlated with distinct autoimmune and allergic disorders, there has been an increasing interest in the possibility that pharmacological manipulation of the Th1/Th2 paradigm could provide novel treatments for human disease. This review summarises the evidence to date, attempts to explain some apparent discrepancies, and indicates opportunities for therapeutic intervention.

Biochemical mechanisms of insulin resistance.

The insulin receptor tyrosine kinase is required for insulin to elicit subsequent biological signalling. Recent studies have identified several endogenous substrates of the insulin receptor kinase, including one called insulin receptor substrate 1 (IRS-1). Tyrosine phosphorylation of this substrate results in its being bound by various proteins containing src homology 2 (SH2) sites, including a phosphatidylinositol 3-kinase and a ras activator complex containing GRB2 and son of sevenless (SOS) 1. Decreases in the insulin receptor tyrosine kinase activity have been observed in various insulin-resistant states, such as non-insulin-dependent diabetes mellitus. A model of insulin resistance has recently been described in which the insulin receptor is expressed in Chinese hamster ovary cells along with the phospholipid- and calcium-activated serine/threonine kinase called protein kinase C. In this model system, activation of protein kinase C is shown to interfere with insulin receptor signalling by inhibiting tyrosine phosphorylation of IRS-1 and its subsequent binding by phosphatidylinositol 3-kinase. Such a model system may be further utilized to determine the detailed biochemical basis for insulin resistance.

Interactions between interleukin-1 and basic fibroblast growth factor on articular chondrocytes. Effects on cell growth, prostanoid production, and receptor modulation.

The interactions of interleukin-1 beta (IL-1 beta) and basic fibroblast growth factor (bFGF) with monolayer cultures of rabbit articular chondrocytes were examined. Both agonists resulted in a synergistic increase in prostanoid production, to levels higher than the maximal level for either protein alone. The synergy was time- and dose-dependent, was augmented in serum-free medium, and was blocked by indomethacin or a polyclonal antibody to IL-1 beta. The proteins had opposite effects on cell growth, and IL-1 beta completely blocked the mitogenic effect of bFGF. Pretreatment of cells with IL-1 beta induced a down-regulation in the number of the bFGF high-affinity receptors. Pretreatment of cells with bFGF increased the number of IL-1 receptors, which was dependent on messenger RNA synthesis.

Biologic effects of an interleukin-1 receptor antagonist protein on interleukin-1-stimulated cartilage erosion and chondrocyte responsiveness.

Recombinant human interleukin-1 alpha (IL-1 alpha) and recombinant human IL-1 beta stimulate matrix proteoglycan degradation and inhibit glycosaminoglycan synthesis in bovine nasal cartilage explants. A 17-kd human recombinant IL-1 receptor antagonist protein (IRAP) caused a concentration-dependent (0.2-200 ng/ml) suppression of the effects of IL-1 alpha and IL-1 beta in cartilage organ cultures. IRAP inhibited the binding of radiolabeled IL-1 alpha to rabbit articular chondrocytes. Matrix metalloproteinase (collagenase, gelatinase, and stromelysin) and prostanoid production by IL-1-activated rabbit articular chondrocytes was also suppressed by IRAP. These results could have potential significance in the development of a new antiarthritis therapy based on an IRAP.

Utilization and formation of amino acids by chicken epiphyseal chondrocytes: comparative studies with cultured cells and native cartilage tissue.

Utilization and production of amino acids by primary cultures of chicken growth plate epiphyseal chondrocytes grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum were investigated in both short-term (6-72 h) and long-term (3-24 day) cultures. Comparative studies were made on levels of free amino acids in chicken blood plasma and serum, and in extracellular fluids from different regions of growth plate cartilage and from two types of muscle. Chondrocytes rapidly consumed glutamine from the medium, and to lesser extents, various other amino acids. In contrast, free ammonia, alanine, glycine, glutamate, proline, and aspartate were released into the medium. The utilization of certain amino acids changed, depending on the stage of culture. Initially glutamate was released into the medium but after confluency was consumed. Conversely, histidine, lysine, and phenylalanine were initially utilized but later were released into the medium. Levels of total free amino acids in extracellular fluids of cartilage and muscle were higher than those in plasma and serum, while in cartilage the levels increased progressively from the resting to the hypertrophic zones. In these sequential regions certain amino acids increased proportionally, whereas others decreased. These interrelationships generally correlated closely with metabolism of amino acids by the cultured chondrocytes. They indicate that significant differences in amino acid metabolism exist between tissue areas and are reflected in the extracellular fluid composition. Accordingly, adjustment of specific amino acids may optimize culture conditions, enabling more normal phenotypic expression in vitro.

Evidence for hybrid rodent and human insulin receptors in transfected cells.

Chinese hamster ovary cells and NIH 3T3 cells overexpressing mutant human insulin receptors were examined for the presence of hybrid receptors composed of human and rodent insulin receptors. In the present studies, most of the endogenous rodent receptors were found to be immunoprecipitated from the transfected cells but not the parental cells with a monoclonal antibody specific for human receptor. These data indicate that in these transfected cells, most of the endogenous rodent receptors are in a hybrid complex with the overexpressed human receptor. These results together with the in vitro studies of Treadway et al. (Treadway, J.L., Morrison, B.D., Soos, M.A., Siddle, K., Olefsky, J., Ullrich, A., McClain, D.A., and Pessin, J.E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 214-218) showing that hybrid receptors exhibit transdominant inhibition explain the prior finding indicating that overexpression of defective insulin receptors interferes with the normal signaling of endogenous receptors.