SHORT COMMUNICATION: Comparison of 5-bromo-2-deoxyuridine and [3H]thymidine for studies of hepatocellular proliferation in rodents

Hepatocyte replication traditionally has been studied by [³H]thymidine(TdR) incorporation into DNA, and more recently using incorporationof 5-bromo-2-deoxyuridine (BUdR), a synthetic analog of thymidinewhich is measured by immunohistochemistry. In studies to compareTdR and BUdR, mice were given the peroxisome proliferator [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (WY-14643) in thediet (0.1%) for 5 days and either TdR (1µg) or BUdR (100mg/kg) on days 2–5. The labeling index (LI) for hepatocytesof WY-14643-treated mice was 4.7% with BUdR and 5.2% with TdR.The LI for control mice was 0.3–0.4% with either label.Partially hepatectomized rats given TdR had a mean LI of 30.0versus 32.0% in rats labeled with BUdR. Sham-operated controlsgiven TdR had a mean LI of 0.2% and BUdR controls had a meanLI of 0.1%. Hepatectomized rats given TdR and BUdR simultaneouslyhad an LI of 20.1% for TdR and 22.4% for BUdR. In these rats,94.1% of the labeled cells contained both markers, whereas 1.9%had only TdR and 4.0% had only BUdR. Comparable labeling indicesusing either TdR or BUdR indicate that the analogs may be usedinterchangeably in short-term in vivo studies of liver cellproliferation.     

A precursor--product relationship exists between oval cells and hepatocytes in rat liver

Oval cell proliferation was induced in twelve male Fischer ratsby administration of 2-acetylaminofluorene (2-AAF) for 2 weeksand by performing partial hepatectomy one week after the beginningof 2-AAF administration. Albumin expression in liver was studiedby using in situ hybridization of ³H-labeled rat albumin riboprobe.Radiolabeled thy-midine was administered to a group of animalsat day 6 after partial hepatectomy. The animals were killedat 0, 3, 7, 9, 11 and 13 days after partial hepatectomy. Bothoval cells and basophilk hepatocytes showed a prominent expressionof albumin, whereas albumin expression hi acidophilic preexistinghepatocytes was decreased. Oval cell nuclei were exclusivelylabeled one day after administration of [³H]thymidine. At day9, 11 and 13 basophilic hepatocytes became labeled and the areaoccupied by these cells increased. This is the first demonstrationof the transfer of radiolabeled thymidine from oval ceDs tonewly formed hepatocytes in vivo. Thus the precursor—productrelationship between oval cells and basophilic hepatocytes hasbeen established.     

Autoradiography of "oval cells" appearing rapidly in the livers of rats fed N-2-fluorenylacetamide in a choline devoid diet

Autoradiographic analysis of liver sections from rats fed thehepatocarcinogen N-2-fluorenylacetamide (FAA) in a choline devoid(CD) diet suggests that proliferating small "oval" cells arisefrom a few small portally-situated cells, and spread rapidlyacross the entire liver lobule. Small cells with detectablegrains are first located where liver plates meet the portalareas. This cell type gradually increases in number over a 10–12day period, then proliferates rapidly. After 28 days, microscopicnodules consisting of heavily labeled large eosinophilic cellsappear, whereas residual hepatocytes are not labeled. Combinedimmunofluorescent and autoradiographic labeling studies revealthat many of the small cells contain AFP; approximately halfof the a-fetoprotein-containing cells are labeled with [³H]thymidine(dT). Feeding CD-FAA diets to rats with hepatocytes prelabeledwith [³H]dT after 70% hepa-tectomy 7 weeks earlier providesdata which suggest that small "oval" cells do not arise fromprelabeled hepatocytes but, instead, infiltrate the prelabeledhepatocytes during the diet induced proliferative phase. Weconclude that "oval" cells arise from a small number of portalcells, not from hepatocytes. Exact identification of the ovalcell precursor is not possible, but it could be a "stem" cell.Although hepatocyte-like properties are found in small cells(e.g., albumin staining), there is no evidence that they differentiateinto normally functioning hepatocytes.     

In vitro labeling and gold activation autoradiography for determination of labeling index and DNA synthesis times of solid tumors.

In vitro labeling and gold activation autoradiography were used to determine the [3H]thymidine ([3H]TdR)-labeling indices and DNA synthesis times for C3H/He spontaneous mammary tumors. Three variations of the [3H]TdR, [14C]thymidine ([14C]TdR) double-labeling method, together with double-emulsion autoradiography, were used to determine the DNA synthesis times (TS). Tumors labeled totally in vivo (in vivo-in vivo method) and tumors labeled with [3H]TdR in vivo and subsequently labeled with [14C]TdR in vitro showed similar TS values. DNA synthesis times for tumors determined totally in vitro by double labeling (in vitro-in vitro method) were significantly longer than those observed in vivo; however, identical samples subjected to Hypaque-Ficoll gradient separation after double labeling showed TS's similar to those found in vivo. Furthermore, the interval between [3H]TdR and [14C]TdR administration had no effect on TS estimates in vitro. Gold activation autoradiography was used in the present experiments to reduce autoradiographic exposure times. This method, together with in vitro labeling, permits [3H]TdR labeling index and TS determinations after 6-hr and 7-day exposures, respectively.     

Reciprocal relationship between development of glutathione S-transferase positive liver foci and proliferation of surrounding hepatocytes in rats

The effects of 2-acetylaminofluorene (2-AAF), pbenobarbital(PB) and butylated hydroxyanisole (BHA) on the competitive proliferationof glutathione S-transferase (GST-P) positive liver cell fociinduced by diethylnitrosamine (DEN) and surrounding hepatocyteswere studied. Rats were given a single i.p. injection of 200mg/kg body wt of DEN and from 2 weeks later were given a dietcontaining 0.02% 2-AAF (group 1), 0.05% PB (group 2), 2.0% BHA(group 3), or no supplement (group 4) for 6 weeks. All ratswere subjected to partial hepatectomy (PH) at the end of week3. Animals from each group were killed 1, 2, 3 or 4 days or1, 2, 3 or 5 weeks after PH. Sequential changes in cellularproliferations after PH were investigated by immunohistochemicalstaining for GST-P and autoradiography of [³H]thymidine. Thedevelopment of GST-P positive foci was enhanced strongly by2-AAF and slightly by PB and was inhibited by BHA. In the grouptreated with 2-AAF, proliferation of hepatocytes adjacent tofoci was almost completely inhibited [labelling index (LI)=0.4 –1.5], but GST-P positive foci cells had a high LI(16.2–26.1) 1 week after PH. In the group treated withPB, proliferation of surrounding hepatocytes was slightly inhibited(LI one day after PH = 16.0) compared with that of control (LI=24.2),but proliferation of cells in GST-P positive foci was not inhibited(LI one day after PH=11.3; control value=11.0). BHA retardedrecovery of the LI (LI 4 days after PH = 8.1; control value,4.0) and did not inhibit proliferation of surrounding hepatocytes.These results indicated an inverse relationship between thedevelopment of GST-P positive foci and proliferation of surroundinghepatocytes.     

The apparent inhibition of urothelial DNA synthesis in neonatal rats by dietary saccharin

[³H]Thymidine ([³H]TdR) incorporation into urothelial DNA of male neonatal rats was measured autoradiographically at birth and during the first 3 weeks of life. The rats were derived from control parents and those fed saccharin (1, 3, 5 and 7.5%) in the diet from before pregnancy. [³H]TdR incorporation was inhibited and there were more lightly labeled cells (compared with controls), in all the saccharin-exposed rats in a rough dose-dependent manner. The results, in comparison with controls, suggest that saccharin exposure in utero causes DNA damage in the neonatal urothelium manifesting as reduced thymidine incorporation and a greater proportion of lightly labeled cells.     

Resistance of A-431 epidermoid carcinoma cells to early membrane changes induced by tumour promoters

The A-431 human epidermoid carcinoma cell line was resistantto 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibition ofepidermal growth factor binding, enhanced incorporation of [³H]cholineinto phospholipids and uptake of ⁸⁶Rb and [³H]2-deoxyglucose.The cells were also resistant to TPA-stimulated release of radioactivecholine derivatives and aracholdonic acid from cells prelabelledwith [³H]choline or [¹⁴C]arachidonic acid, respectively. TheA-431 cells did not metabolise [³H]TPA. Despite their TPA-unresponsiveness,A-431 cells contained specific, high affinity binding sitesfor [³H]phorbol-12,13-dibutyrate with characteristics similarto other cultured cell lines.     

Differential responses of nascent DNA synthesis and chain elongation in V79 and V79/79 cells exposed to u.v. light and chemical mutagens

DNA repair after u.v., N-methyl-N-nitrosourea (MNU) and ethylmethanesulphonate (EMS) in Chinese hamster V79 cells and the mutagensensitive derivative V79/79 was investigated by measurementof five parameters: production of strand breaks in templateDNA, incorporation of [³H]TdR, semi-conservative and repairsynthesis, molecular weights of pulse labelled DNA after mutagenexposure (nascent synthesis) and molecular weights of DNA pulselabelled and chased after mutagen exposure (elongation and ligation).Equal template strand breakage was evident in both cell linesimmediately after MNU and EMS exposure and by 4–5 h afterMNU the extent of fragmentation was greater in V79/79 cells.After u.v. irradiation template fragmentation was evident inV79/79 but not in V79 cells, even though V79/79 cells failedto excise cyclobutane dimers and repair synthesis was demonstablein V79 cells but not in V79/79 cells after exposure to all threemutagens. The rate of incorporation of [³H]TdR during semi-conservativeDNA synthesis was inhibited equally in a dose dependent mannerafter u.v. and MNU exposure; incorporation by V79/79 cells wasinhibited to a greater extent than by V79 cells after EMS exposure.Nascent DNA synthesis was suppressed more in V79/79 cells thanin V79 cells after u.v. but to similar extents in both celllines after MNU and EMS treatment. Pulse chase experiments indicateda lower rate of elongation of nascent DNA in V79/79 cells afterMNU and u.v. exposure but little difference was detectable afterEMS.     

Isolation and preliminary characterization of u.v.-sensitive mutants from the human cell line EUE

Five u.v. light-sensitive clones were isolated in the EUE cellline by means of a modified form of the original 5-bromode-oxyuridine(BUdR)-light method worked out by Puck and Kao for the isolationof nutritional mutants. A cell population was mutagenized withethylmethanesulfonate. After the expression time, cells wereu.v.-irradiated and incubated with BUdR to label excision patchesin repair proficient cells. A subsequent irradiation with ‘black’light caused DNA strand breakage in BUdR-substituted cells.During BUdR treatment, hydroxyurea and a fluorochrome (Hoechst33258) were added to possibly enhance the analogue incorporationinto DNA and to increase the photolability of BUdR containingsequences, respectively. Out of 192 colonies selected with thismethod, 38 were isolated and tested for their u.v.-sensitivity.Five of them showed significant, reproducible differences withrespect to the parental line. As a partial characterization,the five u.v.-sensitive clones were assayed for unscheduled[³H]thymidine incorporation after exposure to u.v. light, bymeans of liquid scintillation spectrometry and autoradio-graphy.In all clones, DNA repair synthesis was significantly decreasedwith respect to the parental line.     

Relationship between the cellular accumulation and the cytotoxicity of S12363, a new vinca alkaloid derivative

Summary S 12363, a new vinca alkaloid derivative, was considerably more cytotoxic to murine L1210 cells and five human tumor cell lines (HL60, HT-29, COLO 320DM, NCI-H460, and PANC-1) than was vincristine (VCR) or vinblastine (VLB). S 12363 bound to tubulin in crude extracts from brain or L1210 cells with an affinity similar to that of VLB and VCR (apparentK _d value: 1.1–1.6, 1.2–1.7, and 0.6–0.8 M, respectively). After 1 h exposure, the accumulation of 20 nM [³H]-S 12363 by L1210 cells was 4-to 18-fold that of [³H]-VLB and [³H]-VCR, respectively. After the cells had been preloaded for 1 h with the labeled drugs and then incubated for 3 h in drug-free medium, 37%–55% of the [³H]-S 12363 was retained by the cells vs 36%–47% of the [³H]-VCR and <6% of the [³H]-VLB. Similar results were obtained for the five human cell lines tested. The accumulation factors (intracellular vs extracellular concentrations) found for [³H]-S 12363 (54- to 167-fold) were significantly higher than those observed for [³H]-VCR (5- to 14-fold) or [³H]-VLB (19- to 41-fold). >90% of the radioactivity extracted from L1210 cells that had been treated with [³H]-S 12363 was recovered as unmodified drug, demonstrating that [³H]-S 12363 was not metabolized by these cells. S 12362, which differs from S 12363 only in the absolute configuration of the asymmetric carbon atom of its -aminophosphonic side chain, was 300 times less cytotoxie, bound to tubulin with a lower affinity (apparentK _d value, 4.9–9.6 m), and was neither accumulated nor retained by the cells. Taken together, these results demonstrate that the potency of S 12363 is due at least in part to its cellular accumulation and retention.     

The role of suppression of DNA synthesis and inhibition of cell cycle progression in cellular sensitivity to alkylation damage

A u.v. sensitive Chinese hamster cell line V79/79 has been shownto be also more sensitive to methyl methanesulphonate (MMS)and nitrogen mustard (HN2) exposure than wild-type V79 cells.A comparison of the effects of the two alkylating agents onDNA synthesis measured by [³H]thymidine (TdR) incorporationinto whole cells and by alkaline sucrose gradient sedimentation¹⁴C-labelled template and of pulse labelled DNA revealed nosignificant differences between the responses of the two celllines. The effects of a range of doses of both drugs on therate of progress through the cell cycle was compared using cytofluorimetry.The more sensitive V79/79 cells failed to show a significantdelay in progress through the cell cycle even at the highestdoses tested (0.2 µM HN2 and 2.0 mM MMS). In contrast,V79 cells showed a marked S phase delay in response to bothHN2 and MMS exposure. The possible relationships between failureto delay cell cycle progression, and cellular sensivity arediscussed.     

Effects of nickel compounds on incorporation of [3H] thymidine into DNA in rat liver and kidney

In vivo incorporation of [³H] thymidine into DNA was determinedin rats at 28 h after partial hepatectomy. Administration ofnickel carbonyl (Ni(CO)₄) at 2 or 4 h before sacrifice inhibited[³H] thymidine uptake into liver and kidney DNA. For example,in rats killed 4 h after i.v. injection of Ni(CO)₄ (2 mg Ni/100g), [³H]-labelling of liver DNA averaged 54 (SE ± 10)%of controls (p<0.05), and [³H]-labelling of kidney DNA averaged53 (SE ± 6)% of controls (p<0.01). Injection of NiCI₂(2 mg Ni/100 g, i.m.) 4 h before death did not significantlyaffect [³H] thymidine uptake into liver DNA, but did inhibit[³H] thymidine uptake into kidney DNA (65 ± 6%, p<0.02).Binding of ⁶³Ni to DNA in liver and kidney of rats killed 4h after injection of ⁶³Ni(CO)⁴or ⁶³NiCl² ranged from 0.3 to2.2 mol ⁶³Ni/mol of DNA nucleotides. Ultracentrifugation ofDNA on alkaline sucrose gradients did not reveal any differencesbetween sedimentation profiles of hepatic DNA from Ni(CO)⁴-treatedrats versus paired control rats.     

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.     

Metabolic activation of the food mutagen Trp-P-1 in endothelial cells of heart and kidney in cytochrome P450-induced mice

Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole) is a carcinogenmetabolized by hepatic cytochrome P4501A (P4501A). This studyshowed that there was a highly selective solvent-resistant bindingof radioactive substance in endothelial cells of heart and kidney1 day following injection of [³H]-Try-P-1 (0.1 or 1.5 mg/kg)in NMRI mice treated with the P450-inducing agent ß-naphthoflavone(BNF). In the heart, the binding was highest in capillariesand coronary vessels. In the kidney, the binding was highestin afferent and efferent arterioles and glomerular and peritubularcapillaries. A corresponding localization of radioactivity didnot occur in corn oil-treated mice injected with [³H]-Trp-P-1.On incubation of heart and kidney slices with [³H]-Trp-P-1,there was a binding of radioactivity in endothelial cells ofBNF-treated mice, but not in corn oil-treated mice. The P4501Ainhibitor effipticine abolished the BNF-induced endothelialbinding of [³H]-Trp-P-1 in vivo and in vitro, whereas the effectsof     

Metabolism and DNA binding of aflatoxicol and aflatoxin B1 in vivo and in isolated hepatocytes from rainbow trout (Salmo gairdneri)

The purpose of this study was to compare the metabolism andDNA binding of aflatoxicol (AFL) with that of aflatoxin B₁ (AFB₁)in vivo and in isolated hepatocytes from Mt Shasta strain rainbowtrout (Salmo gairdneri). Maximum total binding of [³H]AFL toliver DNA from trout exposed by intraperitoneal injection was38–47% of that of [³H]AFB₁ over a 1–7 day period.The average AFL/AFB₁ DNA binding ratio in 1-h incubations withisolated hepatocytes was 0.67±0.36 (n=13). In freshlyisolated hepatocytes, substantial interconversion between AFB₁and AFL via reductase and dehydrogenase enzymes was observed.Total in vivo excretion of conjugates in bile over 4 days wasgreater for [³H]AFL substrate than for [³H]AFB₁. To determineif AFL binding was due to direct activation or to prior metabolismto AFB₁ followed by activation, AFL with a tritium atom on thecarbon containing the cyclopentenol function ([1-³H]AFL, wassynthesized and incubated with hepatocytes. Binding of [1-³H]AFLwas 3% that of [³H]AFB₁ and represents only direct binding ofthe intact cyclopentenol epoxide molecule before transformationto AFB₁ and consequent loss of ³H. H.p.l.c. analysis of DNAhydrolyzed after incubation with [1-³H]AFL resulted primarilyin production of non-radioactive 8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyaflatoxinB₁ (AFB₁-N⁷-guanine). A radioactive peak estimated to be 1%as abundant as the AFB₁-N⁷-guanine was also observed. The overallbinding of generally labeled [³H]AFL to trout liver DNA in vivoand in freshly prepared hepatocytes correlates well with availabletumor incidence and mutagenicity data. Conclusions from thesefindings are that direct interaction of AFL-8,9-epoxide withDNA is of relatively minor quantitative importance in rainbowtrout hepatocytes and that the major adduct results from conversionof AFL to AFB₁ prior to epoxide formation.     

Labeled 1,N6-ethenoadenosine and 3,N4-ethenocytidine in hepatic RNA of mice given [ethyl-1,2-3H or ethyl-1-14C] ethyl carbamate (urethan)

Injection of a single dose of [ethyl-1,2-³H] or [ethy1-1-¹⁴C]-ethylcarbamate into 12-day old male [C57BL/6 x C3H/He]F₁ mice orof [ethyl-1,2-³H]ethyl carbamate into adult male A/Jax miceresulted in the formation of labeled 1,N6-ethenoadenosine and3,N⁴-ethenocytidine adducts in the hepatic RNA. These adductswere characterized by comigration on h.p.l.c. of ³H or ¹⁴C inenzymatic hydrolysates of the RNA with synthetic standards.Both the ethenoadenosine and ethenocytidine were further characterizedby their conversion to acetylated products that comigrated withacetylated synthetic standards. The ethenoadenosine was alsoconverted by anhydrous trifluoroacetic acid to a product thatcomigrated with synthetic 1,N⁶-ethenoadenine. The levels ofadducts in the hepatic RNA 12 h after a single injection of0.5–0.6 mg of ethyl carbamate/g body weight were 6–10and 2–3 pmol/mg RNA of ethenoadenosine and ethenocytidine,respectively. No labeled ethenoadenosine or ethenocytidine couldbe detected in the hepatic RNA of mice given [1-¹⁴C]ethanol,an enzymatic hydrolysis product of ethyl carbamate. These dataindicate that ethyl carbamate may be metabolically activatedby dehydrogenation to vinyl carbamate and subsequent epoxidationof the latter compound as previously proposed. Vinyl carbamateepoxide may form etheno derivatives in a manner analogous tothat demonstrated for chloroethylene oxide, an electrophilicmetabolite of vinyl chloride. Vinyl carbamate has been shownto have the same spectrum of tumor induction as ethyl carbamatebut to be much more active than the latter carcinogen.     

HeLa cells resistant to bromodeoxyuridine and deficient in thymidine kinase activity

Mutant sublines of HeLa S3 cells resistant to growth inhibition by bromodeoxyuridine (BUdR) have been isolated. The resistant cell lines (HeLa BU-10, HeLa BU-15, HeLa BU-25, HeLa BU-50, and HeLa BU-100) proliferated in the presence of 10, 15, 25, 50, and 100 μg/ml BUdR, respectively. Extracts from HeLa BU-25, HeLa BU-50, and HeLa BU-100 cells exhibited 2–5 per cent of the thymidine-³H, deoxyuridine-³H, and bromodeoxyuridine-³H phosphorylating activities of parental HeLa S3 cells. HeLa BU-10 and HeLa BU-15 cell extracts were also deficient in thymidine kinase activity, yielding approximately 43 per cent and 8 per cent, respectively, the thymidine kinase activity of parental HeLa S3 cells. The deficiency in thymidine kinase activity of HeLa BU-100 cells was not due to negative feedback inhibition by high levels of BUdR or to interference with the thymidine kinase assay by inhibitors or competing enzymes in the HeLa BU-100 cell extracts. Following 5 weekly passages in media lacking BUdR, the HeLa BU-100 cells did not exhibit increased thymidine kinase activity. Moreover, mixtures of extracts from HeLa S3 and HeLa BU-100 cells displayed a thymidine kinase activity equivalent to the sum of the activities of extracts prepared, respectively, from the HeLa S3 and HeLa BU-100 cells. Radioautographic studies have shown that after HeLa S3 cells were incubated for 6 hours with thymidine-³H, 35–45 per cent of the nuclei were heavily labeled with radioactivity. However, fewer HeLa BU-100 cells displayed labeled nuclei and the nuclei were only lightly labeled. HeLa BU-100 cell extracts contained normal amounts of thymidylate synthetase, thymidylate kinase, and uridine kinase activities. Following infection by vaccinia virus, high levels of thymidine kinase activity were induced in HeLa BU-100 cells.     

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.     

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.