Differential dose-dependent effects of alpha-, beta-carotenes and lycopene on gap-junctional intercellular communication in rat liver in vivo

In order to examine the relevance of alteration of gap-junctional intercellular communication (GJIC) to chemopreventive activity against carcinogenesis, the effects of alpha- and beta-carotene as well as lycopene, typical chemopreventive carotenoids, on cell coupling via gap junctions in rat liver in vivo were studied using a direct functional dye-transfer technique. We found that all three test compounds given at a dose of 50 mg/kg-body weight (b.w.) daily, 5 times by gavage, inhibited GJIC, while similar treatment with 5 mg/kg b.w. caused enhancement, especially in the beta-carotene- and lycopene-treated groups. At the dose level of 0.5 mg/kg b.w., the three compounds had no effect. The findings show that all three agents differentially modulate GJIC depending on the dose, with beneficial effects on cell communication only detected at the one dose. The result suggests that determination of the dose of chemicals to be used is crucial for human intervention studies.     

Selective inhibition of primary acute myeloid leukaemia cell growth by lovastatin

The insulin receptors from erythrocytes of 50 patients with non-insulin-dependent diabetes mellitus were tested for their ability to autophosphorylate. The assay was performed by a new enzyme-linked immunosorbent assay system that used monoclonal anti-insulin receptor antibodies absorbed to microtiter plates as a first antibody and polyclonal antiphosphotyrosine antibody as a labeled second antibody. By this assay, 3 patients were identified with defects in their insulin receptor kinase, although their defects appeared heterogeneous. Patient 1 had 85% less maximal autophosphorylation with a normal ED50 (1.6 x 10(-9) M insulin). Patient 2, who had polycystic ovary disease, had a 49.2% decrease in maximal autophosphorylation of insulin receptors, and the ED50 was shifted to the right (5.6 x 10(-8) M). Patient 3 with acanthosis nigricans had a normal maximal autophosphorylation, but the ED50 shifted to the right (2.9 x 10(-8) M). The mechanisms for the diversity detected in this assay is not known, but this technique has sufficient specificity and sensitivity to be used to screen for insulin-resistant patients who have a lack of kinase activity.     

Carotenoids and intercellular communication via gap junctions

Long-term cultured murine embryonic yolk sac cells that are capable of forming capillary structures when cultured on base membrane proteins (Matrigel) were successfully transfected with a human growth hormone antagonist (G120R) gene. Cells that stably express relatively high levels of G120R were co-implanted s.c. with Matrigel into BALB/c mice. G120R can be detected in the sera of those implanted mice for more than 14 days at levels from 4 ng/ml to 28 ng/ml. The insulin-like growth factor-1 levels in the sera of those implanted mice were significantly affected by the delivered G120R. One of the physiological effects of G120R delivered by this murine embryonic yolk sac cell-derived mini-organ system is to decrease the growth rate of the implanted mice. This gene delivery system can also be used as an alternative to transgenic animals to study protein function in vivo.     

Reversal of indomethacin-induced inhibition of tubuloglomerular feedback by prostaglandin infusion

Experiments were performed in rats to study the effect of infusion of PGI2, PGE2, and PGF2 alpha on tubuloglomerular feedback responses (i.e. the change of SNGFR in response to a change of loop of Henle flow rate) in the presence and absence of simultaneous inhibition of endogenous PG synthesis with indomethacin. Infusion of PGI2 or PGE2 at rates that did not alter arterial blood pressure did not significantly modify the magnitude of feedback responses (PGI2 8.5 micrograms/hr, PGE2 85 micrograms/hr). Some inhibition of feedback responses was seen when PGI2 and PGE2 were administered at higher rates that were associated with a reduction of blood pressure (PGI2 20 micrograms/hr, PGE2 200 micrograms/hr). PGI2 (8.5 micrograms/hr) and PGE2 (85 micrograms/hr) largely prevented feedback inhibition induced by indomethacin. When given subsequent to indomethacin PGI2 and PGE2 restored feedback responsiveness almost to normal. In contrast, PGF2 alpha did not influence feedback inhibition caused by indomethacin. Infusion of PGI2 induced partial restoration of feedback responses in DOCA-salt treated animals in which the feedback system is virtually completely inactive. Our results indicate that availability of PGI2 or PGE2 is necessary for the normal operation of the tubuloglomerular feedback mechanism for control of nephron filtration rate.     

Downregulation of the pro-apoptotic protein Bak is required for the ras-induced transformation of intestinal epithelial cells

Anoikis is a form of programmed cell death induced in normal epithelial cells by detachment from the extracellular matrix [1] [2] [3]. In epithelial cells of the intestine and other organs, activated rasinduces resistance to anoikis [3] [4], but the actual molecular effectors directly involved in the apoptotic machinery that execute or block anoikis have not yet been identified. Bak, a pro-apoptotic member of the Bcl-2 family, is downregulated in a high proportion of colorectal tumours [5]. In addition, Bak is an important regulator of apoptosis in normal intestinal epithelial cells [6] [7]. Here, we show that activated rasinduces the downregulation of Bak in rat and human intestinal epithelial cells. This ras-induced downregulation of Bak expression could be suppressed by an inhibitor of phosphatidylinositol (PI) 3-kinase, an enzyme already implicated in ras-induced resistance to anoikis [8]. Ectopic expression of Bak in ras-transformed rat intestinal epithelial IEC-18 cells inhibited ras-induced resistance to anoikis and significantly reduced their tumorigenicity. We conclude, therefore, that the ability of rasto downregulate Bak, and the consequent resistance to anoikis, are essential components of the transforming capacity of this oncogene in intestinal epithelial cells.     

Regulation of connexin-43 gap junctional intercellular communication by mitogen-activated protein kinase

Activation of the Ras/Raf/mitogen-activated protein kinase kinase/mitogen-activated protein (MAP) kinase signaling cascade is initiated by activation of growth factor receptors and regulates many cellular events, including cell cycle control. Our previous studies suggested that the connexin-43 gap junction protein may be a target of activated MAP kinase and that MAP kinase may regulate connexin-43 function. We identified the sites of MAP kinase phosphorylation in in vitro studies as the consensus MAP kinase recognition sites in the cytoplasmic carboxyl tail of connexin-43, Ser255, Ser279, and Ser282. In this study, we demonstrate that activation of MAP kinase by ligand-induced activation of the epidermal growth factor (EGF) or lysophosphatidic acid receptors or by pervanadate-induced inhibition of tyrosine phosphatases results in increased phosphorylation on connexin-43. EGF and lysophosphatidic acid-induced phosphorylation on connexin-43 and the down-regulation of gap junctional communication in EGF-treated cells were blocked by a specific mitogen-activated protein kinase kinase inhibitor (PD98059) that prevented activation of MAP kinase. These studies confirm that connexin-43 is a MAP kinase substrate in vivo and that phosphorylation on Ser255, Ser279, and/or Ser282 initiates the down-regulation of gap junctional communication. Studies with connexin-43 mutants suggest that MAP kinase phosphorylation at one or more of the tandem Ser279/Ser282 sites is sufficient to disrupt gap junctional intercellular communication.     

Reversal by trypsin of the inhibition of active transport by colicin E1

The time course for inhibition of proline transport and irreversible loss of cell viability after treatment with colicin E1 was measured as a function of temperature between 13 and 33 degrees C, using a thermostatted flow dialysis system. Complete inhibition of proline transport at 33 and 13 degrees C occurred in 0.5 min and 3 to 5 min, respectively, after addition of colicin E1 at an effective multiplicity of about 4. At these times, the fractional cell survival, assayed by dilution directly from the flow dialysis vessel into trypsin, ranged from 35 to 80%, with viability always greater than 50% at the lower incubation temperatures. Further studies were carried out at 15 degrees C. Complete inhibition of proline transport, which required 2 to 3 min, occurred much more rapidly at 15 degrees C than did the decay of trypsin rescue, which required 10 to 15 min to reach a survival level of 10 to 20%. The direct addition of trypsin to the flow dialysis vessel, after an addition of colicin E1 that caused complete inhibition of proline or glutamine transport, resulted in restoration of net transport. The restored level was typically about 40% of the control rate, and was very similar to the fractional cell viability measured after incubation in trypsin in the same vessel. It is concluded that trypsin can restore active transport to a significant fraction of a cell population in which transport has been initially inhibited by colicin E1.     

Reversal of somatostatin inhibition of insulin and glucagon secretion

These studies were designed to elucidate the mechanism of inhibitory action of somatostatin (SRIF) on glucagon (IRG) and insulin (IRI) secretion. Studies were carried out in the unrecirculated isolated rat pancreas perfusion with arginine 19.2 mM and glucose 5.5 mM as stimulus primarily for IRG but also IRI secretion. The effects of excess Ca++ (15.2 mEq./L.) and excess K+ (12.8 mEq./L.) on IRG, IRI, and the SRIF-inhibited pancreas were studied. Ca++ excess in five perfusions strikingly stimulated IRG secretion (+92 per cent) but only stabilized IRI secretion compared with control perfusions. K+ excess (in seven perfusions) markedly inhibited IRG secretion (-39 per cent) while stimulating IRI secretion (+16 per cent). Restoration of normal concentrations of K+ resulted in a rebound of IRG to levels 120 per cent that of controls. SRIF, at concentrations from 0.1-20 ng./ml., produced inhibition of both IRG and IRI. In 11 perfusions, with SRIF at 10 ng./ml., IRG decreased more than IRI (-75.2 per cent IRG and -46.9 per cent IRI). In five perfusions, addition of Ca++ (15.2 mEq./L.) 10 minutes after SRIF was started resulted in a reversal of IRG inhibition to 69.4 per cent and IRI to 73.2 per cent of the arginine controls. The reversal by Ca++ of SRIF effect on IRG was greater at higher concentrations of Ca++, suggesting some form of competition. In four perfusions, excess K+ reversed SRIF-induced IRI inhibition to 79.6 per cent that of controls but had no effect on IRG inhibition. Studies in vitro with isolated islets revealed that SRIF (2 mug./ml.) inhibited 45Ca uptake of islets as did epinephrine (10(-5) M). It was concluded that SRIF-induced inhibition of hormone release appears related to an action on Ca++ uptake.     

Stimulation of gap junctional intercellular communication by thalidomide and thalidomide analogs in human skin fibroblasts

It has been speculated that gap junctional intercellular communication (GJIC), an intercellular signalling pathway, is involved in embryogenesis by coupling compartments of the same developmental potential. We found that thalidomide induces GJIC in human fibroblasts after activation by liver microsomes in concentrations as low as 10(-7) M. Treatment of cells with the thalidomide analog EM-12 increased GJIC without prior activation. No alteration of GJIC was detected with phthalimide and glutamate, the components of thalidomide. However, 2-phthalimido glutaric acid (PGA), a hydrolysis product of thalidomide, stimulated GJIC without activation at concentrations between 10(-10) M and 10(-5) M. We suggest modification of GJIC as a biochemical mechanism responsible for pharmacological and toxicological properties of thalidomide and related compounds.     

Wounding alters epidermal connexin expression and gap junction-mediated intercellular communication

We show that connexin expression and in vivo patterns of communication were dramatically altered in response to epidermal wounding. Six hours after injury, Cx26 was up-regulated in the differentiated cells proximal to the wound, but was down-regulated in cells located at the wound edge. In contrast, Cx31.1 and Cx43 were down-regulated in cells both peripheral to and at the wounded edge. These patterns of altered connexin expression were detectable as early as 2 h after wounding and were most pronounced in 24-h old wounds. Increased expression of Cx26 was still evident in the hyperproliferative epidermis of 6-day old wounds. In vivo dye transfer experiments with Lucifer yellow and neurobiotin confirmed that junctional communication patterns were altered in ways consistent with changes in connexin expression. The data thus suggest that intercellular communication is intimately involved in regulating epidermal wound repair.     

Rhabdomyolysis, acute renal failure and hepatopathy induced by lovastatin monotherapy

The immunoglobulin VH gene rearrangement in a primary cutaneous, large-cell (centroblastic and immunoblastic) B-cell lymphoma was analyzed using a micromanipulation/single-cell polymerase chain reaction technique. In all single B cells obtained from CD20-stained skin sections that gave a polymerase chain reaction product (eight of 27 in biopsy I), the same VHDJH rearrangement, consisting of DP-54-DIR1-JH3a genes, was detected, with no intraclonal nucleotide diversity. Comparison with the most closely related germline counterpart showed significantly altered complementarity determining gene regions as a result of somatic mutations, suggesting an antigen-driven selection and expansion ofthis particular B-cell clone. Interestingly, in a biopsy obtained from the patient 9 mo later, during disease progression (deep muscle infiltration), the lymphoma cells again contained the same VHDJH gene rearrangement (six of 18 in biopsy II) without any further somatic mutations. Therefore, it is suggested that the cutaneous lymphoma characterized throughout this study descended from postgerminal center B-cells.     

Gap junctional intercellular communication of primary and asbestos-associated malignant human mesothelial cells

We examined gap junctional intercellular communication (GJIC) of primary human mesothelial cells and cell lines of asbestos-associated human pleural mesotheliomas, and the effect of asbestos and other mineral fibres on these cells. In homologous cultures, the GJIC capacity of six out of seven tumour cell lines was markedly less than for primary mesothelial cells. This defect in GJIC appeared not to be at the expression level of mRNA and protein of the gene encoding the 43 kDa gap junction protein. In heterologous cocultures of tumour cells and primary mesothelial cells, however, 80-90% of the tumour cell/normal cell contacts were functional. Exposure of primary mesothelial cells to TPA, a phorbol ester tumour promoter, resulted in marked inhibition of GJIC, being an action common to numerous tumour promoters. Such an effect though was not observed with the carcinogenic mesothelioma-inducing mineral fibres chrysotile and amosite, neither with glass wool. These results suggest that a permanent defect in GJIC capacity is a common feature of human mesothelioma cells, but how mineral fibres are involved in the process of mesotheliomagenesis is still unclear.     

Disruption of gap junctional intercellular communication in human renal cancer cell lines

OBJECTIVES: Gap junctional intercellular communication (GJIC) is believed to play an important role in the maintenance of cell homeostasis, and its disruption may be associated with carcinogenesis. However, GJIC has not been detected in many human cancers. We therefore studied the regulation of GJIC in human renal cancer cell lines. METHODS: We examined the human renal cancer cell lines, ACHN and NT, as well as Madin-Darby canine kidney (MDCK) cells as a positive control, using GJIC assays, Northern blotting to detect connexin 43 mRNA, immunofluorescent staining, and Western blotting of connexin 43 protein. RESULTS: GJIC of ACHN and NT was completely blocked. In ACHN cells, connexin 43 mRNA was not altered. However, connexin 43 protein was aberrantly localized and phosphorylated connexin 43 protein had disappeared. Both connexin 43 protein and its mRNA were undetectable in NT cells. CONCLUSIONS: GJIC in human renal cancer cell lines is impaired and various pathways may inhibit this mechanism in renal cancer. We believe that connexin plays an important role in renal carcinogenesis.     

Distinct signaling pathways regulate transformation and inhibition of skeletal muscle differentiation by oncogenic Ras

Expression of oncogenic Ras in 23A2 skeletal myoblasts is sufficient to induce both a transformed phenotype and a differentiation-defective phenotype, but the signaling pathways activated by oncogenic Ras in these cells and their respective contribution to each phenotype have not been explored. In this study, we investigated MAP kinase activity in control 23A2 myoblasts and in 23A2 myoblasts rendered differentiation-defective by the stable expression of an oncogenic (G12V)Ha-ras gene (Ras9 cells). The MAP kinase immunoprecipitated from Ras9 cells was 30-40% more active than that from control 23A2 cells. To establish if this elevated MAP kinase activity is essential to the maintenance of the oncogenic Ras-induced phenotype, we utilized the selective MAP kinase kinase 1 (MEK1) inhibitor, PD 098059. PD 098059 decreased the MAP kinase activity in Ras9 cells to the level found in 23A2 cells. PD 098059 did not affect the ability of 23A2 myoblasts to differentiate. PD 098059 reverted the transformed morphology of Ras9 cells but did not restore the ability of these cells to express the muscle-specific myosin heavy chain gene or to form muscle fibers. Treatment with PD 098059 also did not affect the ability of oncogenic Ha-Ras to establish a non-myogenic phenotype in C3H10T1/2 cells co-expressing MyoD. These results demonstrate that the activation of MAP kinase is necessary for the transformed morphology of Ras9 cells but is not required by oncogenic Ras to establish or to maintain a differentiation-defective phenotype. While these data do not rule out the possibility that constitutive signaling by MEK1 or MAP kinase could inhibit myoblast differentiation, they clearly demonstrate that other pathways activated by oncogenic Ras are sufficient to inhibit differentiation.     

Preservation of endogenous antioxidant activity and inhibition of lipid peroxidation as common mechanisms of antiatherosclerotic effects of vitamin E, lovastatin and amlodipine

OBJECTIVES: We sought to document the common mechanisms of the antiatherogenic effects of the cholesterol-lowering hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin, the dihydropyridine Ca2+ blocker amlodipine and the antioxidant vitamin E. BACKGROUND: Vitamin E, HMG-CoA reductase inhibitors and Ca2+ blockers each inhibit atherosclerosis in hypercholesterolemic animals. METHODS: New Zealand White rabbits were fed regular chow (Group A), chow with 1% cholesterol (Group B), 1% cholesterol diet plus lovastatin (Group C), 1% cholesterol diet plus vitamin E (Group D) or 1% cholesterol diet plus amlodipine (Group E) for 12 weeks. The extent of aortic atherosclerosis was measured by planimetry of the sudanophilic area. Malondialdehyde (MDA) and superoxide dismutase (SOD) in blood were measured as indexes of lipid peroxidation and antioxidant activity, respectively. RESULTS: Group A rabbits showed no atherosclerosis, whereas Group B rabbits had 17.4 +/- 9.3% (mean +/- SD) of the aorta covered with atherosclerosis, and Groups C, D and E rabbits had significantly less atherosclerosis. Plasma SOD activity was lower in Group B than in Group A (6.9 +/- 1.1 vs. 12.8 +/- 1.5 U/ml, p < 0.01) and was preserved in the groups given lovastatin, vitamin E or amlodipine with a high cholesterol diet. The serum MDA level was higher in Group B rabbits than Group A rabbits (12.1 +/- 2.6 vs. 1.2 +/- 0.1 nmol/ml, p < 0.01) and increased minimally in rabbits given lovastatin, vitamin E or amlodipine with a high cholesterol diet. In in vitro experiments, both lovastatin and amlodipine preserved SOD activity and reduced the oxidizability of low density lipoproteins by rabbit leukocytes. CONCLUSIONS: This study suggests that a reduction in lipid peroxidation and preservation of SOD may be common mechanisms of antiatherosclerotic effects of lovastatin, vitamin E and amlodipine.     

Social facilitation and inhibition of emotional expression and communication.

Does the presence of others facilitate or inhibit emotional expression? Female "senders" (n?=?45) viewed 12 emotionally loaded slides either alone or with another sender while responses were secretly videotaped. In Study 1, 14 "receivers" guessed the type of slide viewed by dyads more accurately (n?=?.366). In Study 2, 42 receivers viewed 10 senders with friends, 10 with strangers, and 10 alone. One dyad member was covered so that only 1 sender was visible. Analysis revealed significant effects of condition (alone, friend, or stranger; n?=?.456), slide type (sexual, scenic, unpleasant, or unusual; n?=?.325), and the Condition?×?Slide Type interaction (n?=?.350). Strangers had overall inhibitory effects on others. Thus, both social facilitation and inhibition of expression occurred on the basis of the emotional stimulus and personal relationship involved. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Connexin expression and intercellular communication in two- and three-dimensional in vitro cultures of human bladder carcinoma

The identification of gap-junctional proteins (connexins) and the preparation of related antibodies provides new tools to study patterns of intercellular communication in tumors. Focusing on the biology of human bladder carcinoma, we compared the expression of gap-junctional proteins (connexins Cx26, Cx32, and Cx43) with a dye-coupling assay for gap-junctional intercellular communication in three cell lines with different urothelial differentiation. The cell lines HCV-29, RT4, and J82 were initially grown as monolayers of different ages. Connexin expression was found mostly positive over the time of culture and found constantly negative only in J82 cells for Cx26 and HCV-29 cells for Cx32. In HCV-29 cells, Cx26 increased in positivity over the time of culture. Western blotting with the antibodies confirmed the findings. Comparisons of dye transfer using Lucifer Yellow showed an increase of coupling in the normal urothelial cell line HCV-29 in contrast to a decrease of coupling in the tumor cell lines. Data were extended by multicellular spheroid (MCS) co-cultures with the stromal fibroblast line N1. In three-dimensional cultures as MCSs, Cx26 was increased in proximity of RT4 tumor cells to fibroblasts, and positivity was maintained in J82 cells. E-cadherin expression in cell lines showed no change in dependence of growth state. The data suggest that Cx26 plays a role in negative growth control or differentiation of urothelial cells. Preliminary comparative data on normal and neoplastic urothelium show all three connexins in normal urothelium, in contrast to varying amounts of Cx43 and low amounts of Cx32 in tumors and evident loss of Cx26 in low-grade tumors. Discrepancies between monolayer and MCS cultures are most likely due to higher differentiation in MCSs, and the continuation of systematic work with heterologous MCSs is indicated for more information on the role of gap-junctional proteins in human tumors.     

Effect of perchloroethylene and its metabolites on intercellular communication in clone 9 rat liver cells

Gap junction intercellular communication (IC) is thought to be important in chemical carcinogenesis as abnormalities in IC have been found in cancer cells. Perchloroethylene (PERC) is metabolized in rodent liver to dichloroacetic acid (DCA) and trichloroacetic acid (TCA), which are rodent liver carcinogens. Chloral hydrate (CH) and trichloroethanol (TCEth) are kidney metabolites. We used Lucifer yellow scrape-load dye transfer as a measure of IC to look at the effect of PERC, DCA, TCA, CH, and TCEth on Clone 9 cell cultures (normal rat liver cells). Four independent experiments were performed for each chemical using exposure times of 1, 4, 6, 24, 48, and 168 h. Concentrations for each chemical varied and were based on preliminary data on effect and cytotoxicity. To compare the relative effectiveness of each chemical to cause biological change, we identified the lowest concentration and shortest time to significantly reduce dye transfer. DCA caused a significant change at 10 mM at 6 h; TCA, 1 mM at 1 h; CH and TCEth, 1 mM at 24 h; and PERC, 0.01 mM at 48 h. Over a 24-h treatment period, the relative efficiencies, as defined by the concentration needed to produce 50% reduction in IC, were PERC (0.3 mM) > TCA (3.8 mM) > TCEth (6.6 mM) = CH (7.0 mM) > DCA (41 mM). Time-course data indicated that PERC, DCA, and TCA produced reduction in IC in a similar fashion, but 5 mM CH or TCEth exhibited variances from these results and may indicate specific cell responses to these chemicals. The mechanism(s) responsible for inhibition of IC by these structurally related chemicals needs to be established.