Tumor promoter 12-O-tetradecanoylphorbol-13-acetate receptors in normal human transitional epithelial cells

As a prelude to study the promotion with TPA of in vitro transformationof human urothelial cells (HUC) in culture, we characterizedtumor promoter TPA receptors in primary cultures of HUC. [³H]TPAbound specifically to intact living HUC; maximum specific bindingwas attained in 30 min at 37°C. [³H]TPA bound to HUC ina saturable and competitive manner. Scatchard analysis of specificbinding to intact cells displayed a single slope correspondingto an equilibrium dissociation constant (Kd) of 0.56 nM; atsaturation TPA-binding capacity was 2.37 pmol/10⁶ HUC (1.43x 10⁶ sites per cell). [³H]TPA bound specifically and with highaffinity to the particulate fractions of HUC; binding was bothsaturable and reversible. Saturation of the specific bindingof [3H]TPA occurred at 1 nM at 4°C. Scatchard analysis ofspecific binding to the particulate fraction displayed a singleslope corresponding to a Kd of 1.08 nM; at saturation TPA-bindingcapacity was 2.05 pmol/mg protein (750 000 molecules per HUC).[³H]TPA binding was inhibited by the biologically active phorbolester, phorbol didecanoate, whereas inactive phorbol did notcompete for TPA binding. Binding was not affected by sodiumsaccharin, epidermal growth factor, retinoic acid or dexamethasone.[³H]TPA bound specifically to the HUC cytosolic fraction butonly in the presence of calcium and phosphatidylserine. Calcium-activatedand phospholipid-sensitive protein kinase activity was detectedin HUC fractions. These results indicate the presence of high-affinityspecific receptors for TPA in HUC.     

Effects of cell surface receptor-altering agents on the binding and biological activity of 12-O-tetradecanoylphorbol-13-acetate in isolated epidermal cells

Isolated epidermal cells were incubated with a variety of compoundsknown to interfere with or alter the ultrastructure of cellsurface receptors, and the ability of 12-O-tetradecanoyl-phorbol-13-acetate(TPA) to bind to these cells and induce epidermal ornithinedecarboxylase (ODC) activity was investigated. The - and ß-adrenergicantagonists, phentolamine and propranolol, and the cholinergicantagonist, atropine, which competed effectively for the bindingreceptors of [³H]dihydro--ergocryptine, [³H]dihydroalprenolol,and [¹⁴C]acetylcholine, did not inhibit the induction of ODCactivity by TPA or the specific binding of [³H]TPA to the cells.Neuraminidase treatments caused a time- and dose- related releaseof sialic acid from the cells and enhanced the stimulatory effectof cholera toxin on basal and TPA-induced ODC activities asmuch as the monosialoganglioside GM₁. Neuraminidase and theother membrane-altering agents, fucosidase, galactosidase, galactoseoxidase, phospholipases A2 and C, and NaIO₄, were used aloneand/or in various combinations in our studies. All treatmentstested inhibited the specific binding of several ¹²⁵I-labeledhormones and epidermal growth factor to the cells. In contrast,none of these treatments was able, in the same cell system,to affect either the binding or the biological activity of TPA.Therefore, these results suggest that the primary interactionof TPA at the plasma membrane level as well as its biologicaleffect in the intact cell do not proceed through adrenergicor cholinergic receptors and do not require the integrity ofthe cell surface glycoconjugates and phospholipids. In addition,the inhibitory effect of retinoic acid on TPA-induced ODC activityremained unaffected by some of the above treatments, suggestingthat retinoic add is unlikely to interfere with TPA interactionsat the plasma membrane level.     

Inhibition by 12-O-tetradecanoylphorbol-13-acetate of 17{beta}-estradiol-induced initiation of DNA synthesis in rabbit endometrial cells in culture

The initial effects of 12-0-tetradecanoylphorbol-13-acetate(TPA) and 17/3-estradiol on initiation of DNA synthesis by primarycultures of estrogen-responsive rabbit uterine epithelial cellswere determined by autoradiography after a [³H]thymidlne pulse.In contrast to the stimulatory response produced by estradiol,TPA (1.6 x 10^-7 M) reduced the fraction of cells synthesizingDNA for up to 48 h. Cells synthesizing DNA at the time TPA wasadded were found to be less sensitive to its inhibitory effect,suggesting that TPA blocked the cells from entering the S phaseof the cell cycle. The cells were unresponsive to estradiolduring the TPA-induced inhibitory period, although TPA did notinterfere with the binding of [³H]estradiol to the hormonalwhole-cell receptors. The results indicated that TPA acted toreduce the fraction of cells initiating DNA synthesis by a mechanismthat appears to be independent of estrogen involvement.     

The effect of 12-O-tetradecanoyphorbol-13-acetate (TPA) on phospholipid metabolism of human epidermal keratinocytes in culture

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)stimulated the release of [³H]choline from prelabelled membranephospholipids of cultured keratinocytes obtained from normalhuman skin. In contrast, TPA in the concentration range of 10^-12to 10^-6 g/ml failed to induce deacylation of [³H]arachidonicacid or stimulate [³H]prostaglandin production in prelabelledkeratinocytes. In addition, TPA did not induce [³H]choline incorporationinto the membrane phospholipids of these cells. The previouslyreported inability of TPA to stimulate a proliferative responsein these cell cultures may be related to the resistance of thesecells to TPA-induced alterations of arachidonate metabolism.     

Formation of a glucuronide conjugate of 12-O-tetradecanoylphorbol-13-acetate by LLC-PK1 renal epithelial cells in culture

The metabolism of [³H]tetradecanoylphorbol-13-acetate [³H]TPA)was studied in LLC-PK₁ cells, a differentiated renal epithelialcell line. In contrast to earlier studies in both fibroblasticand epithelial cells, a major metabolite of [³H]-TPA by thesecells was a new, previously unobserved species which was muchmore hydrophilic than TPA or its major metabolite in other cells,phorbol-13-acetate. Incubation of culture medium containingthis metabolite with ß-glucuronidase greatly reducedthe amount of this polar metabolite, accompanied by a nearlyequal increase in the amount of [³H]-TPA extractable by organicsolvents from this medium. In several experiments this TPA-glucuronicacid conjugate accounted for 20–50% of the total metabolitespresent after several days of incubation of LLC-PK1 cells with[³H]TPA. Two other polar renal epithelial cell lines, derivedfrom canine (MDCK) and bovine (MDBK) kidney, were incapableof converting [³H]TPA to a ß-glucuronidase sensitiveproduct. A time course study indicated that once formed, theTPA-glucuronic acid conjugate was stable in LLC-PK₁ cells. Incubationof LLC-PK₁ conditioned medium containing the conjugate withother cell types such as primary hamster embryo cells or primarynewborn hamster epidermal cells indicated that this materialwas stable in these cultures also. However, primary culturesof rat hepatocytes, and to a lesser extent a human hepatomacell line, were capable of further metabolism of this conjugateto a form insensitive to ß-glucuronidase treatment.This is the first evidence for the formation of a TPA-glucuronicacid conjugate either in vivo or in vitro, and while it is stablein the presence of the cells which produce it, this compoundcan also be further metabolized, presumably via ß-glucuronidasein at least one cell type (i.e., hepatocytes). The biologicalsignificance of the formation and cleavage of this TPA-glucuronicacid conjugate remains to be determined.     

Retinoic acid nuclear receptors and tumor promotion: decreased expression of retinoic acid nuclear receptors by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

During studies to determine the mechanism of tumor promotionby 12-O-tetradecanoylphorbol-13-acetate (TPA), we found thatTPA downregulates mouse epidermal retinoic acid nuclear receptors(RAR), a superfamily of nuclear steroid/thyrold receptors implicatedin mediating effects of retinoic acid (RA). Application of TPAto mouse skin decreased the binding of [³H]RA to RAR from mouseepidermal nuclear extracts. In this experiment, 20 nmol of TPAwas applied to mouse skin and 3.5 h later binding of [³H]RAto RAR was analyzed by chromatography on a size-exclusion column.TPA treatment resulted in an     

Comparative effects of a complete tumor promoter, TPA, and a second-stage tumor promoter, RPA, on intercellular communication, cell differentiation and cell transformation

The biological activities in vitro of the incomplete (second-stage)tumor promoter, 12-O-retinoyl phorbol-13-acetate (RPA), andthe complete tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate(TPA) were compared. The doses of TPA and RPA necessary to inhibitthe specific binding of [³H]-phorbol-12,13-dibutyrate ([³H]PDBu)to BALB/c 3T3 cells (50% inhibition doses; ID₅₀; 8–13ng/ml) were very similar; however, RPA was less potent thanTPA in inhibiting [³H]-PDBu binding to Friend erythroleukemiacells (FELC). Intercellular communication between BALB/c 3T3cells, measured by transfer of microinjected fluorescent dye(Lucifer Yellow), was inhibited by RPA as well as by TPA; TPAwas about five times more potent than RPA. RPA also inhibitedFELC differentiation induced by hexamethylene bisacetamide (HMBA)but not the differentiation of a TPA-resistant clone. The dose-responsesof these two compounds in inhibiting differentiation of bothTPA-sensitive and resistant FELC were very similar. When TPAand RPA were compared in their promoting activity of in vitrocell transformation of BALB/c 3T3 cells initiated with 3-methykholanthrene(MCA, 0.1 µg/ml), both TPA and RPA significantly increasedthe yield of morphologically transformed foci, and RPA was 10times more potent than TPA. These results suggest that RPA andTPA share many common in vitro biological effects and that thesein vitro studies do not allow us to delineate clearly the effectof a second-stage tumor promoter from that of complete tumorpromoters such as TPA.     

Characterization of a human placental factor which inhibits specific binding of phorbol esters to cultured cells

Phorbol ester receptors have been demonstrated in a varietyof cells and tissues using [³H]phorbol-12,13-dibutyrate (PDBu)as a ligand. In a search for possible endogenous ligand(s) forthe receptor, we used the human placenta as a source. A factorthat can inhibit the binding of [³H]PDBu on different typesof cells was purified (133-fold) from an extract of human placenta.This factor, PEBIF (‘phorbol ester binding inhibitoryfactor’), is sensitive to pepsin and resistant to trypsintreatment. It is heat- and acid (pH3)-stable and can be precipitatedby 80% ethanol with no loss of activity. PEBIF inhibits bindingwhether it is added before or after incubation of [³H]PDBu withhuman amniotic membrane cells (FL). Inhibition occurs at both37?C and 4?C and is rapid and reversible; it does not requireintact cells, since it also occurs with membrane fractions.PEBIF does not act Llke a binding protein for PDBu, and thekinetics of the inhibition on FL cells is non-competitive. Inhibitionwas also observed in rat liver cells (IAR 6) and Friend erythroleukaemiacells (FELC). Differentiation of FELC induced by hexamethylenebisacetamide can be inhibited by 12-O-tetradecanoyl-phorbol-13-acetate(TPA) only if TPA-sensitive cells (TS 19–101) are used;no inhibition is observed with TPA-resistant cells (TR 19–9).The same is true of PEBIF. It has been shown that these twoclones have about the same number of receptors, with no changeof affinity; and the extent of inhibition of PDBu binding byPEBIF was similar in the two clones. Like TPA, PEBIF can increase2-deoxyglucose uptake in mouse fibroblasts (BALB/3T3 cells).These data suggest that this physiological factor may play arole in the regulation of cell differentiation and/or in themodulation of carcinogenesis.     

Specific binding of the tumor promoter TPA in various mouse organs as measured by a 'cold acetone-filter assay'

Rapid, specific, saturable and partially reversible bindingof the tumor promoter 12-O-tetradecanoylphorbol-13-acetate ([³H]TPA)to a particulate fraction of mouse brain can be demonstratedby means of a ‘cold acetone-filter assay’; by washingwith cold acetone at -20°C, nonspecific binding of the highlylipophilk [³H]TPA to membranes can be reduced to 10% of thetotal binding. A comparative study of homogenates of severalorgans with this test revealed specific [³H]TPA binding/ µgDNA in the order brain > lung spleen Over kidney thymus ovaries. Specific binding sites were also detected hi 600 xg supernatant fractions of homogenates from epidermis, forestomach,glandular stomach and small intestine. Competition experimentsshowed displacement of [³H]TPA by TPA > phorbol-12,13-didecanoate> 12-deoxyphorbol-13-tetradecano-ate > phorbol-12,13-dibutyrate(PDBu) > phorbol-12,13-diacetate > 4-O-methyl-TPA >4-phorbol-12,13-didecanoate in the brain particulate fraction.Specific [³H]TPA binding is sensitive to heat, periodate anddithiothreitol, but is relatively insensitive to urea or to1–5% solutions of several common detergents. It is estimatedthat the present binding test is 500 times more sensitive thanthe widely-accepted [³H]PDBu assay; perhaps more important,the present method employs TPA, which is extremely effectiveboth as a tumor promoter in vivo and as a pleiotropic effectorof cells in vivo and in vitro.     

Specific binding of phorbol esters to Friend erythroleukemia cells -- general properties, down regulation and relationship to cell differentiation

Specific and saturable binding sites for [20-³H]phorbol 12,13-dibutyrate([³H]PDBu) were demonstrated in intact Friend erythroleukemiacells (FELC), in which inducible erythroid differentiation isreversibly inhibited by phorbol esters. The binding of [³H]PDButo intact cells was maximal within only 15 min of incubationat 37°C, after which there was a gradual decrease; bindingat 4°C however, was a slow process, requiring > 180 minfor maximal binding. A Scatchard analysis showed that the dissociationconstant for binding of [³H]PDBu is 8.3 nM; at saturation, 1.75x 10⁵ molecules of [³H]PDBu are bound per cell. The bindingof [³H]PDBu is blocked by 12-O-tetradecanoyl phorbol-13-acetate,phorbol 12,13-didecanoate, mezerein, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and resiniferatoxin, but not by phorbol or4-phorbol 12,13-didecanoate. There was, in general, a good correlationbetween the potency of these agents in inhibiting [³H]PDBu bindingand their activity in promoting tumors on mouse skin. Inducersof differentiation, such as hexamethylene bisacetamide, dimethylsulfoxide and butyric acid, as well as inhibitors of cell differentiation,dexamethasone and local anesthetics, did not significantly blockthe binding of [³H]PDBu to intact FELC. When FELC were inducedto differentiate with 4 mM hexamethylene bisacetamide (80% ofcells were benzidine-positive), a slight decrease (10–20%)in the number of binding sites at saturation was seen, but thedissociation constant was not changed. When the cells were preculturedwith non-radioactive phorbol esters, a significant decreasein [³H]PDBu binding was observed, suggesting a homologous downregulation of phorbol ester receptors. Scatchard analysis indicatedthat the decrease in [³H]PDBu binding was due to a decreasein the number of binding sites and not to a change in affinity.Such specific phorbol ester binding sites might mediate a numberof biochemical and biological effects of phorbol esters on FELC.     

Phorbol esters stimulate the rapid release of choline from prelabelled cells

Evidence implicating the cell surface membrane as the primarysite of action of phorbol ester tumor promoters has led us tocharacterize early changes in phospholipid metabolism whichoccur in response to these compounds. When C3H10T mouse embryofibroblasts were incubated with [³H]choline for 24 h, {smalltilde}78% of the cell associated material was found in phosphatidylcholine and the remainder in sphingomyelin and the acid solublepool. Within 5 min of exposure of these prelabelled cells tothe potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate(PPD) the release of water soluble [³H] metabolites from cellsinto the medium was enhanced 2-fold and by 60–120 minthe release was 2–5 times that found in control cultures.The released material was identified by chromatography as cholineand phosphoryl choline. Evidence was obtained that this materialwas not derived exclusively from the acid soluble pool or fromthe degradation of labelled sphingomyelin. Choline metaboliterelease was concentration dependent between 10^–8 and 10^–7M TPA and was not associated with cell toxicity. Phorbol-12,13-didecanoate(PDD) was also active but 4PDD, which is not a tumor promoter,was inactive. Neither cyclohexlmide (4–40 µg/ml)nor cordycepln (4–40 µg/ml) blocked the TPA inducedrelease. The release was, however, temperature sensitive anddid not occur at 4°C. TPA did not induce the release of[³H]inositol from prelabelled phosphatidyl inositol. Although,the calcium ionophore A23187 [GenBank] induced the release of arachidonicacid from prelabelled cells it did not induce choline release.In addition, 5,8,11,14-eicosatetraynoic acid, which inhibitsboth lipoxygenase and cyclooxygenase activity, did not inhibitTPA stimulation of choline release. We propose that the bindingof TPA to cell surface receptors leads to a rapid activationof membrane associated phospholipase(s) that specifically degradephosphatidyl choline. This effect may be analogous to the abilityof other agonists to activate degradation of phosphatidyl inositol.     

The down-modulation of receptors for phorbol ester tumor promoter in primary epidermal cells

The specific [20-³H]phorbol 12,13-dibutyrate ([³H]PDBu) bindingto intact epidermal cells displayed the phenomenon of down-modulation,i.e., the specific binding of [³H]PDBu to its receptors on primaryepidermal cells reached a maximum within 1 h and steadily declinedthereafter (Proc. Natl. Acad. Sci. USA, 78, 2549, 1981). Theapparent down-modulation of radiolabel resulted from a partialloss in the total number of receptors; the affinity of receptorsfor the ligand was essentially unchanged. A number of agentssuch as chloroquine, methylamine, or arginine which are knownto prevent clustering, down-modulation, and/or internalizationof several hormone receptors did not affect the down-modulationof phorbol ester receptors. Furthermore, cydoheximide had noeffect either on down-modulation or on the binding capacityof cells. The surface binding capacity of down-modulated cellsfollowing a 90-min incubation with unlabeled ligand was almostreturned to normal within 1 h. This recovery did not dependon protein synthesis, as it was insensitive to cydoheximide.However, the surface binding capacity of epidermal cells wassensitive to the serum. Cells maintained for more than 3.5 hat 37°C in the serum-free medium showed a loss of specific[³H]PDBu binding; no such loss was seen in medium containingserum. The effect of the antidepressant drug chlorpromazine,which is known to interact with calmodulin, on [³H]PDBu bindingwas also investigated. The cells pretreated with chlorpromazineshowed a significant decline in their cell binding capacityfor [³H]PDBu; this was due to a decrease in the number of availablebinding sites, because the affinity of binding remained almostunchanged. Our data indicate that the effect of chlorpromazineon [³H]PDBu binding is probably unrelated to its calmodulin-bindingactivity.     

The transport of reactive intermediates in a co-cultivation system: the role of intercellular communication

The transport of reactive intermediates through gap junctionswas studied in a co-cultivation system consisting of primarychick-embryo liver cells and V79 Chinese hamster cells. Theformation of gap junctions was studied by measurement of theincorporation of [³H]hypoxanthine in HGPRT deficient V79 mutantcells after co-cultivation with the hepatocytes. Under controlconditions the heterologous gap junctions allowed for the transferof [³H]hypoxanthine resulting in an average value of 13 grains/cell.Addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate(TPA), inhibited this transfer in a dose related way to backgroundvalues. The transfer of reactive intermediates was measuredas the number of sister chromatid exchanges (SCEs) induced inthe V79 cells. For both compounds tested, benzo[a]pyrene anddimethylnitrosamine, transport of reactive intermediates throughgap junctions was observed. When the inhibitory effects of TPAwere investigated at different time points after start of theco-cultivation, inhibition was measurable after 6 h and increasedto a maximal inhibition of 50%, observed after 24 h. No effectof TPA was observed on the number of SCEs. When the metaboliccooperation deficient V79 mutant cell line MC^–-27, wasused the effects were completely comparable to those of TPAfor both compounds. The lower mutagenic effects in the MC^–-27cells cannot be attributed to a lower intrinsic sensitivityfor mutagens of these cells.     

Lysines of histone 1 represent the principal target for covalent binding of microsomally activated benzo[a]pyrene in vitro

The principal target amino acid residue for covalent bindingof metabolically activated [³H]benzo[a]pyrene (B[a]P) to histone1 (H1) has been identified. Highly purified calf thymus ³H wasmodified by incubation with [³H]B[a]P in the presence of ratliver microsomes. The relative distribution of [³H]B[a]P versusspecific amino adds among the N-bromosuccinimide peptides ofH1 suggested that lysine may be the target. This was testedby selectively blocking the nudeophilic amino groups of lysineby amidination prior to [³H]B[a]P binding. Increasing levelsof amidination of H1 resulted in corresponding decreases inthe reactivity of this histone toward [³H]B[a]P. Amidinationof 93% of the lysines blocked 87% of the [³H]B[a]P binding indicatingthat lysine was the principal target. N-terminal amino acidsequencing of [³H]B[a]P-modified H1 confirmed that lysine residueshad bound [³H]B[a]P.     

Identification of phorbol ester binding activity in the particulate and soluble fractions of human fetal tissues

The presence of specific binding sites for phorbol esters wasdemonstrated in various human fetal tissues by binding assaysof [³H]12-O-tetradecanoylphorbol-13-acetate (TPA) to particulateand cytosol fractions. Maximal binding activity of the solublereceptor is dependent on calcium and phosphatidylserine. Markeddifferences in receptor levels were found in various human fetaltissues. However, all the tissues tested contained binding sitesfor TPA. Particulate receptor concentration was highest in thebrain, while the cytosolie receptor concentration was high inbrain, stomach and liver. Human fetal tissues bound TPA witha K_d between 10 and 30 nM.     

The effect of phorbols on metabolic cooperation between human fibroblasts

Autoradiography has been used to study the effect of 12-O-tetradecanoylphorbol-13-acetate(TPA), 4-O-methyl TPA, and phorbol on metabolic cooperationbetween human diploid fibroblasts. When the donors, hypoxanthine-guaninephosphoribosyl transferase + (HGPRT+) cells, and recipients,HGPRT– cells, were plated together in the presence of[3H]hypoxanthine and either 4-O-methyl TPA or phorbol, nearlyall interactions that developed in 4 h were positive for metaboliccooperation whereas when high concentrations of TPA were used,the number of positive interactions was significantly less thanthe control. If the phorbol analogs were added after the donorsand recipients had made contact, the number of positive interactionswas the same as the control in all cases. However, althoughprimary recipients in the cultures that had been treated withphorbol had the same number of grains as those in the control,primary recipients in cultures that had been treated with TPAor high concentrations of 4-O-methyl TPA had significantly fewergrains than those in the control. TPA treatment for 4 h hadno effect on total [³H]hypoxanthine incorporation or incorporationinto acid-soluble and acid-insoluble fractions. Thus, the effectof TPA on metabolic cooperation is interpreted as a reductionin the transfer of [³H]nucleotides and is an indication of aninterference with intercellular communication.     

Biotransformation of aflatoxin B1 in human lung

In addition to being a potent hepatocarcinogen, aflatoxin B₁(AFB₁) is a pulmonary carcinogen in experimental animals, andepidemiological studies have shown an association between AFB₁exposure and lung cancer in humans. This study investigatedAFB₁ bioactivation and detoxification in human lung tissue obtainedfrom patients under-going clinically indicated lobectomy. [³H]AFB₁was bioactivated to a DNA binding metabolite by human wholelung cytosols in a time-, protein concentration-, and AFB₁ concentration-dependentmanner. Cytosolic activation of [³H]AFB₁ correlated with lipoxygenase(LOX) activity and was inhibited by the LOX inhibitor nordihydroguaiareticacid (NDGA; 100 µM), indicating that LOXs were largelyresponsible for the observed cytosolic activation of AFB₁. Inwhole lung microsomes, low levels of indomethacin inhibitableprostaglandin H synthase (PHS)-mediated [³H]AFB₁-DNA bindingand cytochrome P-450 (P450)-mediated [³H]AFB₁-DNA binding wereobserved. Cytosolic glutathione S-transferase (GST)-catalyzeddetoxification of AFB₁–8,9-epoxide, produced by rabbitliver microsomes, was minimal at 1 and 10 µM [³H]AFB₁.With 100 µM [³H]AFB₁, [³H]AFB₁–8, 9-epoxide conjugationwith reduced glutathione was 0.34 ± 0.26 pmol/mg/h (n= 10). In intact, isolated human lung cells, [³H]AFB₁ bindingto cellular DNA was higher in cell fractions enriched in macrophagesthan in either type II cell-enriched fractions or fractionscontaining unseparated cell types. Indomethacin produced a 63–100%decrease in [³H]AFB₁-DNA binding in macrophages from five ofseven patients, while NDGA inhibited [³H]AFB₁ -DNA adduct formationby 19, 40 and 56% in macrophages from three of seven patients.In alveolar type O cells, NDGA decreased [³H]AFB₁-DNA bindingby 30–100% in cells from three patients and indomethacinhad little effect. SKF525A, an isozyme non-selective P450 inhibitor,enhanced [³H]AFB₁ binding to cellular DNA in unseparated cells,macrophages, and type II cells, suggesting that P450-mediatedbioactivation of AFB₁ is not a major pathway by which AFB₁–8,9-epoxideis formed in human lung cells. Overall, these studies suggestthat P450 has a minor role in the bioactivation of AFB₁ in humanlung. Rather, LOXs and PHS appear to be important bioactivationenzymes. Co-oxidative bioactivation of AFB₁, in combinationwith the low conjugating activity displayed by human lung cytosolicGSTs, likely contributes to human pulmonary susceptibility toAFB₁.     

Inhibition by palmitoylcarnitine of adhesion and morphological changes in HL-60 cells induced by 12-O-tetradecanoylphorbol-13-acetate

Effects of DL-palmitoylcarnitine (PC), an inhibitor of calciumactivated, phospholipid-dependent protein kinase (protein kinase C), on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell differentiation were investigated in human promyelocytic leukemia cells (HL-60). TPA caused HL-60 cell adhesion concomitant with morphological changes, and an increase in acid phosphatase activity. The median effective concentration was 1 nM, which corresponded well to the dissociation constant of [3H]TPA binding to the cell extract. [3H]TPA binding to the cell extract was saturable and reversible. The maximal number of [3H]TPA-binding sites was 1.5 pmol/mg protein and a Hill coefficient was unity, indicating noncooperative interactions. PC, but neither palmitic acid nor DL-carnitine, inhibited the TPA-induced cell adhesion and morphological changes with the median inhibitory concentration of 1 microM, whereas a TPA-induced increase in acid phosphatase activity was not affected by 3 microM PC. Addition of PC 1 or 2 days after the addition of TPA was also effective in inhibiting the cell adhesion. Among various acylcarnitines, PC had the largest effect. [3H]TPA binding to the cell extract was not inhibited by PC at the concentration which was effective in inhibiting the TPA-induced cell adhesion. These results indicate that protein kinase C possibly mediates HL-60 cell differentiation induced by TPA.     

Role of intercellular communication in the promotion of C3H/10T1/2 cell transformation

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) uponintercellular communication and promotion were studied in culturesof C3H/10T1/2 mouse embryo fibroblasts. Cell-to-cell communicationwas quantitated by autoradiographic analysis of [³H]uridinetransfer from prelabelled donor cells to an excess of unlabelledrecipient cells during a 4 h co-cultivation. Extensive transferof label was observed from donor to recipient cells in contact.Treatment of non-transformed C3H/10T1/2 cultures with 250 ng/mlTPA at co-cultivation of donor and recipient ceils markedlyinhibited intercellular communication during the 4 h incubation,producing an 80% reduction in undine exchange relative to solvent-treatedcontrol cultures. Concentrations of TPA ranging from 0.25 to25 ng/ml were also effective in inhibiting [³H]uridine exchangein a dose dependent fashion from 13% to 74%. This inhibitionof intercellular communication was transient; cells exposedto 250 ng/ml TPA for 1.5 h prior to co-cultivation with TPAexhibited a 60% inhibition and the exchange of undine had increasedto control values in cultures pretreated for 12–72 h.An examination of label transfer between non-transformed andtransformed C3H/10T1/2 cells indicated that both the extentof inhibition by TPA and the kinetics of communication inhibitionwere similar to that observed for non-transformed cells. Initiationand promotion experiments demonstrated that exposure to 250ng/ml TPA for 5 weeks, but not to reduced concentrations of0.25, 2.5 or 25 ng/ml, was capable of promoting morphologicaltransformation. The lack of correlation between the dose responsesof TPA for promotion and for reduction of cell-to-cell communication,and the transient nature of intercellular communication inhibitionby TPA, suggests that an inhibition of cell-to-cell communicationis not a sufficient event for promotion of oncogenic transformationin these cells.     

Precursor-product relationship between oval cells and hepatocytes: comparison between tritiated thymidine and bromodeoxyuridine as tracers

The expansion and differentiation of oval cells in the acetylaminofluorene(AAF)/partial hepatectomy (PH) model was studied utilizing pulse-chaselabeling with both tritiated thymidine ([³H]TdR) and bromodeoxyuridine(BUdR). Animals in which a significant decrease in serum albuminand increase in alanine aminotransferase and bilirubin wereobserved demonstrated the most prominent differentiation ofoval cells into hepatocytes. Administration of [³H]TdR or BUdR,either individually or together, to the animals on day 6 afterpartial hepatectomy resulted in labeling of the majority ofthe oval cells by days 7 and 9 after PH. A striking differencein the distribution of [³H]TdR- and BUdR-labeled cells in thedouble labeling experiments was observed on day 11, at whichtime the number of [³H]TdR-labeled cells increased 6-fold andthat of double labeled cells decreased 2-fold. Furthermore,on day 11 the basophilic foci were weakly positive for BUdRand negative at later time points in animals receiving BUdRalone or together with [³H]TdR. In contrast, the cells in basophilicfoci as well as transitional cells were positive for [³H]TdR.Cells heavily labeled with both [³H]TdR and BUdR were presentat all time points, indicating an inhibition of the proliferativeactivity. Pulse labeling of rat liver epithelial cells withBUdR in vitro demonstrated that immunodetection of BUdR waslost after three or more cell divisions. We conclude that theBUdR tagging method is particularly sensitive to label dilutionduring cell cycling and may not be suitable for establishmentof a precursor-product relationship between cell lineages whenthe progenitor population proliferates more than three times.