Differences in the calcium-mediated regulation of gap junctional intercellular communication between a cell line consisting of initiated cells and a carcinoma-derived cell line

Differences in calcium-mediated regulation of gap junctionalintercellular communication (GJIC) between a cell line consistingof mouse epidermal initiated cells (3PC) and a mouse epidermalcarcinoma-derived cell line (CA3/7) were studied. Under lowextracellular calcium (Ca²⁺_e) conditions (0.05 mM) CA3/7 cellsshowed a low level of GJIC compared with 3PC cells. High Ca²⁺_e(1.20 mM) raised GJIC between CA3/7 cells to the GJIC levelof 3PC cells, which in turn remained unchanged under these conditions.Raising the free intracellular calcium concentration (Ca²⁺₁),using a calcium ionophore (ionomycin) or the Ca²⁺-ATPase inhibitorthapsigargin under low Ca²⁺_e conditions, did not affect theGJIC level between 3PC cells, and increased GJIC between CA3/7cells. Intracellular calcium chelation in 3PC cells under lowCa²⁺_e conditions by ethylene glycol-bis(ß-amino-ethylether) N, N, N', N'-tetra-acetic acid acetoxy-methyl ester (EGTA-AM)decreased GJIC in this cell line. High Ca²⁺_e conditions protectedboth cell lines from a decreased GJIC by EGTA-AM exposure. Inhibitionof calmodulin (CaM) by calmidazolium (CDZ) or N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide(W-7) under low Ca²⁺_e conditions, inhibited GJIC in 3PC cellsand increased GJIC in CA3/7 cells. Inhibition of Ca²⁺/CaM-dependentprotein kinase (Ca²⁺/CaM-PK) by 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine(ML-7) decreased GJIC in both cell lines. Western analysis showedthat Cx43 was more phosphorylated in both cell lines in concurrencewith different effects on the GJIC level. Under conditions inwhich GJIC was inhibited, a decreased immunostaining of Cx43on the plasma membrane was found. The level of immunostainingof the cell adhesion molecule E-cadherin on the plasma membranesof both cell types remained unchanged under conditions in whichGJIC was changed by modulaters of (Ca²⁺)₁, CaM activity, orthe Ca²⁺/CaM-PK activity. These results indicate that differencesexist between 3PC cells and CA3/7 cells in the GJIC regulationby intracellular calcium and calmodulin.     

Inhibition of gap junctional blockage by palmitoyl carnitine and TMB-8 in a rat liver epithelial cell line

Exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) has beenshown to inhibit gap junctional intercellular communication(GJIC) in many cell types in vitro. Using a scrape loading/dyetransfer technique, TPA was shown to cause a dose-dependentand transient inhibition of GJIC in WB-F344, a normal rat liverepithelial cell line. Such a down-modulation of intercellularcommunication was found to be associated with an increase inprotein kinase C (PKC) activity. Translocation of this activityto the particulate fraction occurred 10 min alter exposure to16 nM TPA and was consistent with the time course needed toinhibit GJIC. After 6 h exposure to TPA, essentially all thePKC activity was lost concurrent with the recovery of communicationin these cells. During this time, the cells also became refractoryto inhibition by further addition of TPA. Blockage of communicationinduced by TPA in WB cells was prevented by treating the cellswith 23 µM palmitoyl carnitine for 1 h or 100 µM8-N,N-(diethylamino)-octyl-3,4,5-trimethoxybenzoate for 30 min.The results indicate that TPA transiently modulates GJIC inWB cells and PKC activation is possibly involved in blockageof communication in these cells.     

SHORT COMMUNICATION: Inhibition of gap junctional intercellular communication and delocalization of the cell adhesion molecule E-cadherin by tumor promoters

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) andbenzoyl peroxide (BoP) on gap junctional intercellular communication(GJIC) and the amount and localization of E-cadherin was studiedin initiated mouse epidermal cells (3PC) and in carcinoma cells(CA3/7) originating from the same cell type. In addition, thelocalization and phos-phorylation of connexin43 was studiedin both cell lines and in primary keratinocytes. GJIC inhibitionby TPA and BoP was stronger in primary keratinocytes comparedwith both cell lines. BoP strongly decreased the amount of E-cadherinprotein and the level occurring in the membranes in both celllines, whereas TPA caused a transloca-tion of E-cadherin fromthe membrane towards the cytosol, without decreasing the totalamount of E-cadherin present. The effect of both tumor promoterson connexin43 phos-phorylation and localization was agent aswell as cell dependent. These results show for the first timethat tumor promoters can decrease the quantity and membranelocalization ofE-cadherin in different cell types.     

Inhibition of gap junctional intercellular communication by tumor promoters in connexin43 and connexin32-expressing liver cells: cell specificity and role of protein kinase C

In this study, we investigated whether the tumor promoters, 12-O- tetradecanoylphorbol-13-acetate (TPA), phenobarbital (PB), and 1,1- bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), inhibited gap junctional intercellular communication (GJIC) in a cell-specific or connexin-specific manner and whether protein kinase C was involved. To do this, we used highly communicating WB-F344 rat liver epithelial cells, which express connexin43 as their predominant gap junction protein, WB-aB1 cells, which are a GJIC-incompetent mutant line of WB- F344 cells and that express connexin43, WB-a/32-10 cells, which are a highly communicating derivative of WB-aB1 cells generated by stable transduction with a connexin32 retroviral expression vector, and primary cultured rat hepatocytes, which express conexin32 predominantly. Treatment of WB-F344 and WB-a/32-10 cells, but not hepatocytes, with TPA inhibited GJIC (assayed by Lucifer Yellow dye microinjection). This inhibition involved protein kinase C because (i) inhibition was prevented by co-treatment of the cells with a specific protein kinase C inhibitor, bis-indolylmaleimide, and (ii) treatment with TPA for 24 h had no effect on dye-coupling in agreement with the downregulation of protein kinase C. TPA also caused the internalization of Cx43-containing gap junctions and the formation of a hyperphosphorylated form of Cx43, Cx43-P3, in WB-F344 cells only, but TPA had no effect on Cx32-containing gap junctions or protein mobility. In contrast, PB inhibited GJIC only in hepatocytes and DDT inhibited GJIC in all three types of cells; bis-indolylmaleimide did not block the effects of either agent. These results indicate that the inhibitory actions of TPA and PB on GJIC are cell-specific rather than connexin- specific and that TPA inhibits connexin43 and connexin32-mediated GJIC through a protein kinase C-dependent mechanism.      

Association of viral oncogene-induced changes in gap junctional intercellular communication and morphological transformation in BALB/c3T3 cells

In order to study the relationship between altered gap junctionalintercellular communication (GJIC) and induction of cell transformationby oncogenes, we transfected six viral oncogenes into BALB/c3T3A31-1-1 cells. BALB/c3T3 cells with v-src, v-ras or polyomamiddle T (PyMT) genes grew in soft agar and formed distincttransformed foci in the absence or presence of a vast excessof non-transfected cells. On the other hand, those with v-myc,v-fos or polyoma large T (PyLT) genes expressed less distinctlytransformed phenotypes (less transformed morphology, highersaturation density than non-transfected counterparts and lessgrowth in soft agar), and did not form distinct foci in coculturewith non-transformed cells. When their homologous GJIC capacitieswere examined by the microinjection/dye transfer assay, no decreasein GJIC was observed in any of the v-onc-transformed cells.Non-transformed and all v-onc-transformed cell lines expressedsimilar levels of connexin 43 mRNA. v-myc-, v-fos- and PyLT-transformedcells, but not v-ras-, v-src- and PyMT-transformed cells wereable to communicate heterologously with non-transformed cells.Tumor promoting phorbol esters strongly inhibited GJIC of non-transformedand all v-onc-transformed BALB/c3T3 cell lines. In coculturesof v-myc-, v-fos- or PyLT-transformed cells with non-transformedBALB/c3T3 A31-1-1 cells, 12-O-tetradecanoylphorbol-13-acetate(TPA) increased the number of transformed foci. However, whenthese v-onc-transformed cells were co-cultured with non-transformedBALB/c3T3 A31-1-13 cells (which lose GJIC at growth confluence,as if TPA had been added), no morphologically transformed fociappeared. These results suggest that factors other than GJICare involved in the suppression of oncogene-transformed cellsby surrounding normal counterparts.     

Effects of peroxisome proliferators and 12-O-tetradecanoyl phorbol-13-acetate on intercellular communication and connexin43 in two hamster fibroblast systems

Effects of 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the hepatic peroxisome proliferators (HPPs) clofibrate, methyl clofenapate (MCP), di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) were studied in 2 gap junctional intercellular communication (GJIC) systems, metabolic cooperation in V79 cells and microinjection/dye transfer in Syrian hamster embryo (SHE) cells and V79 cells. TPA inhibited GJIC in both systems but was considerably more potent in V79 cells. SHE cells showed a rapid and transient inhibition of GJIC after exposure to HPPs, with maximal inhibition occurring at 5–15 min. The transient inhibition could be caused by metabolization of the compounds. Clofibrate and MEHP produced strong inhibition of metabolic cooperation in V79 cells at high concentrations, while the effect of MCP and DEHP was lower. However, DEHP, MEHP and clofibrate strongly inhibited dye transfer in V79 cells after a 30 min exposure. Clofibrate also showed a dose- and time-dependent effect on dye transfer in V79 cells. The phosphorylation status of the gap junction protein connexin43 (Cx43) changed minimally in SHE cells after exposure to TPA or HPPs. Cx43 from V79 cells was strongly affected by TPA, but not by HPPs. Immunofluorescence of Cx43 disappeared in both cell types when they were exposed to TPA and MEHP, but not to the other HPPs. Thus, there is no direct correlation between the inhibition of GJIC and changes in the phosphorylation status of Cx43 or the appearance of Cx43 in immunofluorescence experiments. The discrepancies may partly be explained by binding of accessory proteins to Cx43. We point out sequences that may be involved in such binding. Int. J. Cancer 73:240–248, 1997. © 1997 Wiley-Liss, Inc.     

12-O-Tetradecanoylphorbol-13-acetate-induced inhibition of gap junctional communication is differentially regulated in a transformation-sensitive Syrian hamster embryo cell line compared to early passage SHE cells

The transformation-sensitive cell-line BPNi was more susceptibleto 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inhibitionof gap junctional intercellular communication (GJIC) than earlypassage Syrian hamster embryo (SHE) cells, while the potencyof TPA to down-regulate EGF-binding was similar in the two celltypes. The kinetics of TPA-induced inhibition of GJIC suggestedthat different mechanisms may operate at high and low TPA concentrations.The initial inhibition after exposure to high TPA concentrations.The initial inhibition after exposure to high TPA concentrationswas followed by a recovery of GJIC. The recovery was much morepronounced in SHE than in BPNi cells. This effect could notbe explained by differences in down-regulation of protein kinaseC. Removal of high TPA concentrations also resulted in a fasterrecovery of GJIC in SHE than in BPNi cells. In addition, althoughforskolin induced a similar protection against the inhibitoryeffect of TPA on GJIC, forskolin restored GJIC blocked by TPAmuch faster in SHE than in BPNi cells. Thus, BPNi cells aremore sensitive to TPA induced inhibition of GJIC than SHE cells,and have reduced capability to recover from down-regulated GJICas compared to SHE cells.     

Increased gap junctional intercellular communication in Syrian hamster embryo cells treated with oxidative agents

The effects of K₂CrO₄, H₂O₂, benzoyl peroxide, menadione, KBrO₃and UV_365nm on gap junctional intercellular communication (GJIC)have been studied in the 12-O-tetra-decanoylphorbol-13-acetate(TPA)-sensitive Syrian hamster embryo (SHE) cell line BPNi.All agents were found to increase the level of GJIC by 50–100%.Also, in early passage SHE cells, a tendency for increased GJICwas found for the oxidative agents studied. Hydrogen peroxidewas used as a model compound in the subsequent studies. Theincrease in GJIC was reversible, and it was not due to an increasednon-junctional permeability. Hydrogen peroxide counteractedthe TPA-induced decrease in GJIC, regardless of whether thecells were exposed to the compounds simultaneously or the cellswere pre-exposed to TPA before addition of H₂O₂. The GJIC enhancementby H₂O₂ was slightly reduced by the addition of the hydroxylradical scavenger dimethylsulphoxide or by the inhibition ofcatalase by amitrole. The cAMP/ protein kinase A system is theonly characterized signal transduction system that is knownto increase GJIC in most cell types. Hydrogen peroxide did notincrease the amount of cAMP (or cGMP) in BPNi cells, while forskolinand a phosphodiesterase inhibitor had to increase the cAMP levelseveral-fold to affect GJIC to the same degree as the oxidativeagents. Some inhibitors of protein kinase A were assayed fortheir ability to inhibit the increases in GJIC caused by H₂O₂and forskolin. Staurosporine inhibited the forskolin-inducedincrease in GJIC, with much less effect on the H₂O₂-inducedincrease. H8, H88 and H89 had less effect than staurosporineon the forskolin-induced increase in GJIC. The results suggestthat the cAMP/protein kinase A system may not be involved inthe increase in GJIC caused by H₂O₂, although this cannot becompletely ruled out.     

Effects of increased expression of protein kinase C on radiation-induced cell transformation

The in vitro oncogenic transformation of C3H 10T1/2 cells byionizing radiation is known to be enhanced by the tumor promoter12-O-tetradecanoylphorbol-13-acetate (TPA). It is also knownthat the activation of protein kinase C (PKC) by TPA is an importantstep in its tumor-promoting effect. In the present study, weexamined the effects of overexpression of a specific isoformof PKC, PKC_ß1 on 7-ray-induced transformation of 10T1/2cells. In addition, the effects of overexpression of PKC_ß1on the malignant phenotype of a previously transformed 10T1/2cell line were also evaluated. Derivatives of 10T1/2 cells thatstably overexpress PKC_ß1 were obtained by transductionwith the retroviral expression vector pMV7 carrying the ratPKC_ß1 cDNA sequence. We found that the parental 10T1/2cells and a control cell line 10T1/2 MV7, which carried onlythe pMV7 vector without the cDNA insert, expressed dose-dependenttransformation frequencies when exposed to 7-rays. On the otherhand, concurrently treated PKC-overexpressing cells that hadan 11-fold increase in enzyme activity (PKC-4 cells) failedto yield any morphologically identifiable foci. Cell lines thatexpressed lower levels of PKC_ß1 were partially resistantto transformation by     

Cellular-oncogene expression in Friend erythroleukemia cells: relationship to differentiation, commitment and TPA effects

Induced differentiation of Friend erythroleukemia cells canbe continuously and reversibly inhibited by phorbol ester tumorpromoters, e.g. 12-O-tetradecanoylphorbol-13-acetate (TPA),for many years, allowing us to study the mechanisms of differentiationand its inhibition by TPA. We previously identified two stepsin the differentiation process, which can be inhibited by TPA,before and after commitment to differentiation. Using permanentlycommitted cells and TPA-resistant variants we examined the roleof cellular oncogenes in Friend cell differentiation control,and their possible modulation by tumor-promoting phorbol esters.We report here characteristic changes in myc, myb and fos mRNAlevels upon induction of differentiation by hexamethylene bisacetamidetreatment, and present evidence that c-myb mRNA decline is onefeature of Friend cell commitment to differentiation. In addition,using our TPA-sensitive and resistant cell lines, we observedthat the hexamethylene bis-acetamide induced pattern of oncogeneexpression is unperturbed by TPA, regardless of whether thecells are differentiating or not.     

Expression and function of connexin in normal and transformed human keratinocytes in culture

We have studied the gap junctional intercellular communication(GJIC) of immortalized and tumourigenic human keratinocyte celllines and of a spontaneously immortalized non-tumourigenic anda highly differentiating keratinocyte cell line (HaCaT) as thecontrol. In homologous cultures, the GJIC capacity of five squamouscell carcinoma-derived cell lines was 1–27% that of theHaCaT cells. Ha-ras-transfected HaCaT cells with tumourigenicpotential and an SV40 DNA-immortalized cell line had markedlyreduced GJIC capacities. Northern analysis and immunohisto-chemistryshowed that connexin (Cx) 43 is the major gap junction proteinexpressed in the communicating cells. They do not express Cx26 or 32. The low or absent communication observed in certaincell lines was due in some to a lack of Cx 43 gene expression,but in others to aberrant localization of the gap junction protein.GJIC of these cell lines, as well as that of primary normalhuman epidermal keratinocytes, was susceptible to 12-O-tetra-decanoylphorbol-13-acetate-mediatedinhibition. Moreover, GJIC of HaCaT cells and their tumourigenicderivatives is Ca²⁺-dependent. These results, when comparedwith those previously obtained for mouse keratinocyte cell lines,reveal that GJIC of human keratinocytes was correlated to thedegree of differentiation and is controlled in a similar wayto that of murine keratinocytes. Aberrant GJIC seems to be acommon feature of human and murine skin carcinogenesis.     

Effect of tumor-promoting agents on density and morphometric parameters of mouse epidermal Langerhans and Thy-l+ cells

Topical application of tumor-promoting agents to the dorsalskin of female SENCAR mice on a twice-weekly basis resultedin a reduction in density per unit area of bone marrow-derivedThy-1⁺ dendritic cells. Activity was observed for well-establishedtumor-promoting doses of promoting agents of several differentchemical types, including 12-O-tetradecanoylphorbol-13-acetate(TPA, diterpene diester), anthralin (dihydroxyanthrone), andn-dodecane (n-alkane). A reduction in density of the same cellswas also observed on the basis of the asialoGM1 lipid as a surfacemarker after TPA treatment. No parallel effect was observedfor epidermal Langerhans (Ia+) cells, the second major epidermalimmunofunctional cell type, except in the case of anthralin,a finding which is consistent with the reported toxicity ofthis agent. The stage 2 promoting agent mezerein was uniquein inducing a consistent increase in Langerhans cell densities,but did not affect the density of Thy-1⁺ cells when appliedfor a prolonged period unless applied following four doses ofTPA. In contrast to the SENCAR strain, the promotion-resistantBalb/c and C56BL/6 strains showed no response with respect toTPA-induced reduction of Thy-1+ cell density. In addition toeffects on density, the above tumor-promoting agents inducedmorphological changes in both Thy-1⁺ and Langerhans cells. Whenthese changes were placed on a quantitative basis by the calculationof shape and area fraction parameters, marked and significanteffects were observed for the above agents, but not for thepartial promoting agent mezerein nor the non-promoting phorboldiester 4-O-methyl-TPA. The effects of TPA were largely blockedby the potent anti-promoting agent flucinolone acetonide, moreover.These findings further support an important role for quantitativeand qualitative alterations in dendritic epidermal cells intumor promotion.     

DNA sequences in human nasopharyngeal carcinoma cells that specify susceptibility to tumor promoter-induced neoplastic transformation

Homologs of the recently described mouse pro genes, that transfersensitivity to promotion of neoplastic transformation by tumorpromoters, have been cloned from a genomic library of the humannasopharyngeal carcinoma (NPC) cell line CNE₂. Both pro-1 andpro-2 homotogs were identified by screening this library withmouse probes, but only the pro-1 homologs were able to confersensitivity to TPA-induced transformation when transferred topromotion-insensitive mouse JB6 cells. This suggests a possiblerole for the putative pro-1 gene in the etiology of human NPC.     

Peroxisome proliferators show tumor-promoting but no direct transforming activity in vitro

The chemically unrelated hypolipidemic drugs, tiadenol, niadenate, and clofibrate have been tested for carcinogenic and tumor-promoting potential in the C3H/10TI/2 C18 cell test system. None of these chemicals were carcinogenic, while both niadenate and clofibrate were active tumor promoters at micromolar concentrations. All 3 drugs induced the differentiation of C3H/10T1/2 C18 cells to adipocytes. This latter finding confirms previously observed effects of the tumor promoter TPA.     

Redox-active chalcogen-containing glutathione peroxidase mimetics and antioxidants inhibit tumour promoter-induced downregulation of gap junctional intercellular communication between WB-F344 liver epithelial cells

Evidence is mounting supporting a role for oxidative stressin the mechanism of tumour promotion in response to agents suchas 12-O-tetradecanoylphorbol-13-acetate (TPA). In this paperwe demonstrate that glutathione peroxidase-mimetic xenobiotics,ebselen, ebselen-glutathione, a-(phenyl-selenenyl) acetophenoneand bis-(4-aminophenyl) telluride (at concentrations between10 µM and 50 µM) all demonstrate protective effectson TPA-induced downregulation of gap-junctional intercellularcommunication (GJIC) between WB-F344 rat liver epithelial cells.These effects were, in each case, diminished if the cells weredepleted of their intracellular glutathione, and potentiatedif glutathione was supplemented into the incubations. Additionally,bis-(4-aminophenyl) selenide and several N-substituted analogues,possessing potent antioxidant activity but being devoid of GSHperoxidase-mimetic activity, demonstrated remedial activityagainst TPA-induced downregulation of GJIC. Structure-activityrelationships between these molecules showed a strong correlationto the oxidation potential of the selenium atom in the compoundas the bis-(4-nitrophenyl)- and bis-(4-cyanophenyl)- derivatives,which possess poor antioxidant capacity and a half-wave redoxpotential well above +1.0 V, did not affect TPA-induced effectson GJIC. Examination of the mechanism of action of these redox-activecompounds demonstrated correlations between their abilitiesto (i) prevent TPA-induced downregulation of GJIC, (ii) abolishthe accumulation of intracellular oxidants and (iii) preventthe hyper-phosphorylation and internalization of connexin 43in the cells. The active compounds were also able to preventthe rapid, TPA-induced translocation of protein kinase C tothe particulate fraction of the cells, without affecting phorbolester binding. These data support a synergistic role for oxidants.and other TPA-dependent responses within the cell in mediatingthe downregulation of GJIC. Such oxidative metabolism may playa role in the control of translocation of protein kinase C fromthe cytosol to membranes in response to TPA within these cells.Despite the nature of the in vitro test system studied, thedata also clarify the molecular basis for a potential anti-tumourpromotive effect of antioxidants, based on established redoxchemistries of several series of structurally-related molecules.     

Modulation of phenobarbitone-induced loss of gap junctional intercellular communication in hepatocyte couplets

Phenobarbitone (PB) produced a dose-and time-dependent decreasein gap junctional intercellular communication (GJIC) (up to25.0 ± 5.3% inhibition) in rat hepatocyte couplets (4h cultures). The effect was reversible and independent of proteinsynthesis. This inhibition was exacerbated (to 53.3 ±5.4% inhibition) by depletion of intracellular glutathione followingpretreatment with diethylmaleate (0.5 µM, 15 min). Inhibitionwas also significantly enhanced by addition of the cytochromeP450 inhibitors SKF 525A (25 µM) and metyrapone (20 nM).In contrast, hepatocyte couplets derived from rats pretreatedwith PB (0.1% w/v in drinking water) for up to 28 days werefully functional regarding GJIC and were found to be refractoryto the effects of PB added in vitro. This, coupled with thelack of effect of p-hydroxy-PB, suggests that an active metaboliteof PB is not involved in the inhibition of GJIC which may, instead,be through an oxidative stress, which is prevented by glutathione.     

Induction of suppressor activity by tumor-promoting phorbol esters in primary cultures of lymph node cells

We have observed the induction of suppressor activity in primarycultures of lymphocytes by the tumor-promoting phorbol ester12-O-tetradecanoylphorbol-13-acetate (TPA). Suppressor activitywas detected as the ability of TPA-treated lympocytes to inhibitproliferation in mixed lymphocyte cul tures (MLC). Inductionof this activity was dependent on the dose of TPA and was maximalalter 12 h of incubation. Neither the induction of the activity,nor the activity itself was inhibited by indomethacin or interleukin2 (IL2). A comparison of addition of TPA directly to the MLC,addition of TPA pretreated cells as participants in the MLCand addition of cells treated with TPA to induce suppressoractivity suggested that the suppressor activity was only oneof the ways that TPA could inhibit proliferation. This suppressoractivi ty may account for some of the reported effects of TPAin immunological systems in vitro and suggests that suppressorcells could play a role in TPA-mediated promotion in vivo.     

The bile acid analog fusidic acid can replace phosphatidylserine in the activation of protein kinase C by 12-O-tetradecanoylphorbol 13-acetate in vitro

Protein kinase C (PKC) is a Ca²⁺- and phospholipid-dependentprotein kinase which binds and is activated by tumor promoterssuch as the phorbol ester 12-O-tetra-decanoylphorbol-13-acetate(TPA). PKC can be activated in vitro by phosphatidylserine (PS)plus either TPA or Ca²⁺. We report here that the bile acid analogfusidic acid can replace the requirement for PS in the activationof PKC by TPA. In addition, fusidic acid can enhance the activationof PKC by Ca²⁺ and PS as well as by TPA and PS. Fusidic acidis an excellent model compound for in vitro studies of the directeffects of bile acids on PKC activity because, unlike many bileacids, it is completely soluble in standard PKC assay mixtures,obviating the exposure of the enzyme and lipid micelles to organicsolvents. The colonic mucosa is exposed to millimolar concentrationsof bile acids, and we find that fusidic acid stimulates PKCactivity in the presence of TPA with a Ka of 350 µM. Thereis substantial evidence that bile acids are endogenous tumorpromoters, and that colon carcinogenesis is influenced by thecomposition of bile acids in vivo. Thus, fusidic acid may bea prototype of bile acids which could mediate tumor promotion,at least in part, by replacing the requirement for PS in theactivation of PKC.     

Phosphatase inhibitors, gap junctional intercellular communication and [125l)-EGF binding in hamster fibroblasts

A number of phosphatase inhibitors (okadaic acid, calyculinA, aluminium fluoride, sodium molybdate, sodium orthovanadate,pervanadate and vanadyl sulphate) were investigated for theireffects on gap junctional intercellular communication (GJIC)and [¹²⁵I-epidermal growth factor (EGF) binding in early passageSyrian hamster embryo cells (mainly fibroblast-like cells) andin V79 Chinese hamster lung fibroblasts. Only pervanadate decreasedGJIC significantly. After the initial pervanadateinduced decreasethe GJIC recovered rapidly. Only pervanadate was able to changethe band pattern of the gap junction protein connexin43 (cx43)in Western blots. Together this may indicate either that thereis a low turnover of phosphate groups in cx43 under basal conditionsor that the putative phosphatases are not sensitive to mostof the phosphatase inhibitors applied. In contrast, pervanadate,orthovanadate and molybdate decreased [¹²⁵I)-EGF binding. 12-O-Tetradecanoylphorbol-13-acetate(TPA) is able to induce the phosphorylation of both cx43 andthe EGF receptor, concomitantly with a decrease in GJIC and[¹²⁵I)-EGF binding. These effects are reversible after removalof TPA. It could be imagined that other phosphatases would acton cx43 and the EGF receptor after the forced phosphorylationof the two molecules. Thus TPA was used to downregulate GJICand [¹²⁵I]-EGF binding and phosphatase inhibitors were appliedin the upregulation phase. Only pervanadate affected the upregulationof GJIC, and pervanadate, orthovanadate and molybdate affectedthe upregulation of [¹²⁵I)-EGF binding. Thus it is not an identicalcomplement of phosphatases that act on cx43 and the EGF receptor.All the downregulating agents are assumed to be phosphotyrosinephosphatase inhibitors.     

Intercellular communication in colonies of Syrian hamster embryo cells and the susceptibility for morphological transformation

The levels of gap junctional intercellular communication (GJIC)were studied in normal, morphologically altered and morphologicallytransformed colonies formed in the Syrian hamster embryo (SHE)cell transformation assay. The colonies were selected from non-exposeddishes or dishes exposed to 12-O-tetradecanoylphorbol-13-acetate(TPA, 0.16 µM), di(2-ethylhexyl)phthalate (DEHP, 77 µM),Naorthovanadate (vanadate, 3.4 µM) or dieldrin (25 µM)for 7 days during colony formation. TPA, DEHP and vanadate inducedincreased frequencies of morphological transformation of colonies.At the same time, TPA and DEHP decreased GJIC in the coloniesby 30% under the conditions used. All categories of colonieswere equally affected. Vanadate did not change the level ofGJIC in any of the categories of colonies compared to unexposedcontrol. Dieldrin strongly suppressed GJIC in all colonies withoutincreasing the frequency of transformation. The compounds affectedGJIC after short-term exposures (4 and 24 h) to cell monolayersrather similarly to that found after long-term exposure to thecolonies. Transformation assays with coexposure of dieldrintogether with the transforming agents vanadate, DEHP or benzo[]pyrenedid not increase transformation frequencies compared to thetransforming agents alone. The GJIC level in all coexposuregroups was similar to that of dieldrin alone. Furthermore, regardlessof whether dieldrin was present or not, removal of vanadate24 h before fixation of the colonies caused a slight decreasein the transformation frequency. The results suggest that: (i)morphologically transformed colonies have the same ability ofintercellular communication as normal colonies; (ii) decreasedGJIC is probably not either sufficient or necessary to inducetransformation of SHE cell colonies; (iii) a decreased levelof GJIC does not necessarily increase the susceptibility ofSHE cells for transformation; and (iv) inhibition of GJIC maynot have an impact on the maintenance of the transformed phenotypeof SHE cell colonies.