Differential effects of 12-O-tetradecanoylphorbol-13-acetate on cultured normal and neoplastic human bronchial epithelial cells

The effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on 10 human lung carcinoma cell lines were compared to those seen on normal human bronchial epithelial (NHBE) cells. TPA (0.1 to 100 nM) did not enhance the clonal growth rate for any of the cell lines. As little as 3 nM TPA induced the NHBE cells to undergo terminal squamous differentiation and thus completely inhibited their proliferation; in contrast, none of the carcinoma cell lines was significantly inhibited at this concentration, and they all continued to proliferate in as much as 100 nM TPA. To determine if this lack of TPA inhibition of clonal growth reflected resistance to TPA induction of terminal squamous differentiation, we measured the ability of TPA to induce cross-linked envelope formation and to increase plasminogen activator activity in four carcinoma cell lines. Cross-linked envelopes were not induced in two lines, and only a small number were induced in the other two lines relative to NHBE cells; plasminogen activator activity was induced in NHBE cells but not in any of the cell lines.     

12-O-tetradecanoylphorbol-13-acetate-induced differentiation of a human rhabdomyosarcoma cell line

The effect of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on proliferation and differentiation of the human embryonal rhabdomyosarcoma cell line RD was investigated. The proliferation of RD cells is drastically and reversibly inhibited by 100 nM TPA. The effect is evident after 24 h of treatment and is maximal after 50-70 h. The reduction of proliferation in treated cells is followed by increased expression of differentiative characters such as a large increase in muscle myosin expression and in the binding of 125I-alpha-bungarotoxin. Moreover TPA induces the appearance of myotube-like structures, which contain bundles of thick and thin myofilaments along with Z bodies. The described effects are not observed if the TPA-containing medium is replaced daily, thus suggesting that these effects might be related to substances secreted by treated cells. The phosphorylation of three proteins is significantly stimulated by TPA within minutes of its administration to RD cells. Although with a different pattern, the stimulation of protein phosphorylation is still clearly detectable after 6 days of incubation with TPA. These results on human rhabdomyosarcoma cells are, to our knowledge, the first evidence for a growth-inhibiting and a differentiative effect of TPA on a solid tumor of mesodermal origin.     

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.     

Genomic 5-methyldeoxycytidine decreases associated with the induction of squamous differentiation in cultured normal human bronchial epithelial cells

The terminal squamous differentiation of epithelial cells involvesa number of cellular changes. However, the mechanisms underlyingthis differentiation process are unknown. The present studydemonstrates that 5-azacytidine, 12-O-tetradecanoylphorbol-13-acetate(TPA) and type ß-transforming growth factor (TGF-ß)significantly diminish the genomic 5-methyldeoxycytidine (5mdC)content of normal human bronchial epithelial cells concomitantlywith the induction of squamous differentiation. 5-Azacytidineor TPA, but not type ß-transforming growth factor,also initiated decreases in the genomic 5mdC content of differentiationnonresponsive human tumor A549 cells. These effects of TPA ortype ß-transforming growth factor occurred at concentrations of 1 nM and 1.2 pM, respectively, which were approximatelyone tenth of k_d values of their specific receptors. Therefore,the decreases in genomic 5mdC induced by these agents were probablymediated, directly or indirectly, by receptor-ligand interactions.     

Subpopulations of human bronchial epithelial cells in culture respond heterogeneously to 12-O-tetradecanoylphorbol-13-acetate (TPA) and other modulators of differentiation

A greater understanding of the processes involved in the controlof proliferation and differentiation should provide insightinto the mechanisms involved in carcinogenesis. Studies wereundertaken to examine the effects of modulators of differentiationon the proliferation, colony forming efficiency (CFE), and cross-linkedenvelope (CLE) formation of human bronchial epithelial (HBE)cells in culture. Treatment for 24 h with a low concentrationof TPA (0.1 ng/ml) induced a 2-fold increase in CLE but littleinhibition of CFE, suggesting that these two endpoints mightbe occurring independently of one another. Continuous culturein a low concentration of TPA (0.1 ng/ml) arrested growth in>99% of the cells, but after 10–14 days, a few colonieswere observed that were resistant to TPA. This TPA resistantsubpopulation occurred at a frequency of <0.1% of the cellsseeded into cultures. Short term treatment (24 h) with fetalbovine serum (FBS; 1–8%) or calcium (0.5–2 mM) resultedin 2–4 fold increases in CLE with no significant changein CFE. Continuous treatment with FBS or calcium for up to 5days produced similar results. These findings suggest that differentsubpopulations of cells exist within cultures of HBE cultures,perhaps at different states of maturation, and that these subpopulationsrespond differently to modulators of differentiation.     

Suppression by herbimycin A of 12-O-tetradecanoylphorbol-13-acetate-induced dissociation of cells and morphological changes in cultured FL and MDCK cells

Human amnion membrane epithelial (FL) and Madin-Darby canine kidney (MDCK) cells formed colonies which consisted of tightly packed cells. A bundle of actin filaments in the cells at the edge of the colonies ran parallel to the outline of these cells. The cells were attached to the substratum by regions which were observed as black solid lines by interference-reflection microscopy. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 hr induced dissociation of and morphological changes in the cells in the colonies. TPA also disrupted the organization of the actin filaments and the attachment of cells to the substratum. Addition of herbimycin A for 24 hr inhibited the TPA-induced dissociation of the cells in colonies. Treatment with herbimycin A also inhibited TPA-induced modulation of the organization of actin filaments and of the adhesion of cells to the substratum. However, herbimycin A did not inhibit the rapid spreading of cells induced by TPA in the absence of serum. These results suggest that herbimycin A inhibits the dissociation of cells and the morphological changes induced by TPA through suppression of the disruption of a bundle of actin filaments and modulation of adhesion of cells to the substratum.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced induction in Epstein-Barr virus early antigen in Raji cells

Retinol, 5 flavonoids, 3 steroids and 7 sweetening agents were studied for their effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced early antigen (EA) of Epstein-Barr virus (EBV) in Raji cells. Concomitant treatment of Raji cells with TPA and retinol showed inhibition of EA induction. Among flavonoids, quercetin resulted in effective inhibition of EA induction by TPA and α-naphthoflavone showed the weakly inhibitory effect. None of the other flavonoids such as rutin, catechin and β-naphthoflavone affected the induction of EBV-EA by TPA. β-Estradiol obviously inhibited EBV-EA induction by TPA, but hydrocortisone did not show any inhibitory effect on it. Glycyrrhetinic acid, steviol, phyllodulcin and perrillartine also showed the remarkable inhibition of EBV-EA induction. On the other hand, glycyrrhizin and stevioside, glycosides of glycyrrhetinic acid and steviol, did not inhibit the induction of EBV-EA by TPA. Some of the inhibitors reported here may be effective on the inhibition of the in vivo tumor promotion by TPA.     

Acute effects of 12-Otetradecanoylphorbol-13-acetate, teleocidin B, or 2,3,7,8-tetrachlorodibenzo-p-dioxin on cultured normal human bronchial epithelial cells

Effects of teleoddin B, 12-O-tetradecanoylphorboi-13-acetate(TPA), phorbol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and2,7-dichlorodibenzo-p-dioxin (DCDD) on normal human bronchialepithelial cell cultures were assessed by quantitation of cellularmorphology, clonal growth (population doublings per day), cross-linkedenvelope (CLE) formation and the enzymatic activities of arylhydrocarbon hydroxylase (AHH), ornithine decarboxylase (ODC)and plasminogen activator (PA). Toxicity was assessed by clonalgrowth assays. Teleocidin B and TPA had similar effects on growth,morphology and enzyme activities. When the cells were incubatedwith TPA or teleocidin B at concentrations of 1–100 nMfor 6 h, RNA synthesis was unaffected, but DNA synthesis decreasedand squamous differentiation, marked by an increase in cellsurface area and cross-linked envelope formation, was increased.TPA and teleocidin B also increased ODC activity in LHC-0 medium(a maintenance medium without epidermal growth factor) but causeda decrease of ODC activity in LHC-4 (a growth medium containingepidermal growth factor). Finally, TPA and teleocidin B eachcaused an increase of PA and a decrease of AHH activities inboth media. Phorbol, a non-promoting analogue of TPA, had noeffect on growth, morphology or biochemical assays. TCDD (100nM) caused a 15% decrease in cell growth when cells were incubatedin LHC-4, and this was accompanied by an increase in cell surfacearea, PA activity, and CLE formation. TCDD caused an increasein AHH and ODC activities when the cells were incubated in eitherLHC-0 or LHC-4 medium. DCDD did not alter cell growth, and itsmorphological and biochemical effects were similar to thoseof TCDD although less marked. In conclusion, results reportedhere are consistent with the hypothesis that an important propertyof some tumor promoters is their ability to induce terminaldifferentiation in normal, non-initiated epithelial cells.     

Transient protection of cultured human cells against antitumor agents by 12-O-tetradecanoylphorbol-13-acetate

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown in cell cultures to enhance the frequency of resistance to methotrexate. However, we found that TPA could also partially protect human KB cells over a short time (72 h) from the cytotoxicity of several antitumor agents, including 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene)-beta-D-glucopyranoside (VP-16), vincristine, mitoxantrone, and methotrexate, but not 1-beta-D-arabinofuranosylcytosine or 5-fluorouracil. The modes of protection were different for methotrexate and VP-16. Protection by TPA was concentration dependent up to 40 nM and could be accomplished by a 2-h incubation of cells with TPA alone prior to drug treatment. This protection disappeared after a 24-h drug-free incubation. TPA-induced protection could not be mimicked by treatment of cells with 1-oleoyl-2-acetyl-glycerol (a stimulator of protein kinase C) or phospholipase C, which increased the cellular content of diacylglycerols. Thus the action of TPA on protein kinase C may not be sufficient to exert protection. Verapamil, a calcium-channel blocker which has been found to circumvent resistance of multiple drug-resistant cells, also circumvented the protective effect of TPA when used with VP-16. The presence of TPA during a 24-h exposure to radiolabeled VP-16 reduced the cellular drug content by about 30%, whereas verapamil enhanced drug content by at least 50% in TPA-treated and untreated cultures. Since substances with some TPA-like activity have been found in foods and in human circulation, the observation of clinical resistance to some compounds may partly be due to a related mechanism.     

Alteration in glycosphingolipid pattern during phorbol-12-myristate-13-acetate-induced cell differentiation in human T-lymphoid leukemia cells

We analyzed the patterns of glycosphingolipids (GSLs) from a line of cells derived from a clone of the human T-cell leukemia cells (CEM) that had been induced to differentiate by phorbol-12-myristate-13-acetate (PMA) into cells with a suppressor-like phenotype. We characterized the differentiation state of the cells by immunofluorescence by using anti-cell surface differentiation-specific monoclonal antibodies (OKT3, OKT4, OKT6, and OKT8). The GSLs were extracted and separated by thin-layer chromatography and the individual bands were quantitated by a dual-wavelength densitometer or by autoradiography of GSLs labeled with [14C]glucosamine and [14C]galactose. Treatment of the CEM cells with 0.16-16 nM PMA for 6 h to 6 days resulted in a dose- and time-dependent increase in the amount of two neutral GSLs [ceramide monohexoside and ceramide dihexoside] and three gangliosides [monosialoganglioside (GM3), sialosylparagloboside, and disialoganglioside (GD3)]. The increase in the neutral GSLs after PMA treatment reached its maximum at 30 h while GM3 peaked at 96 h. The increases in GM3 and sialosylparagloboside are presumably due to an increase in their synthesis levels because PMA promoted an elevated incorporation of glucosamine and galactose into these GSLs. The increase in the amount of GD3, on the other hand, is due to either a decrease in its degradation or use in other metabolic pathways because no detectable increase in glucosamine and galactose incorporation into this ganglioside could be found. Incubation of control or PMA-induced CEM cells with GM3 fractions purified from either CEM cells, human brain, or dog erythrocytes caused a reduction in cell growth and prevented the increase in reactivity of the induced cells with the OKT3 antibody. Incubation with semisynthetic ceramide dihexoside, however, prevented the decrease in reactivity with the OKT4 antibody. The observed changes in GSL patterns during PMA-induced differentiation of the CEM cells into suppressor-like cells and the inhibition of CEM cell growth by GM3 fractions suggest that the GSLs play a role in the control of cell growth and differentiation in the PMA-treated CEM cells.     

Activated Ki-Ras suppresses 12-O-tetradecanoylphorbol-13-acetate-induced activation of the c-Jun NH2-terminal kinase pathway in human colon cancer cells.

Although the frequency of activated Ki-ras genes is high in human colorectal tumors, much less is known of activated Ki-ras-mediated signaling pathways. Using gene targeting, we examined HCT116 cells that contain the Gly-13-->Asp mutation of Ki-ras and activated Ki-ras-disrupted clones derived from HCT116. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced immediate early genes, such as c-Jun, c-Fos, and Egr-1 in activated Ki-ras-disrupted clones, whereas c-Jun induction was rare in HCT116. TPA induced both phosphorylation of stress-activated protein kinase kinase 1 (SEK1) and c-Jun NH2-terminal kinase (JNK) in the activated Ki-ras-disrupted clones but not in HCT116. On the other hand, TPA-induced mitogen-activated protein kinase kinase 1/2 (MEK1/2)-extracellular signal-regulated kinase (ERK) activation was equally induced between HCT116 and the Ki-ras-disrupted clones. Furthermore, TPA-induced SEK1-JNK activation was observed in a DLD-1-derived activated Ki-ras-disrupted clone but not in DLD-1. The TPA-induced SEK1-JNK activation in these disrupted clones was completely inhibited by the protein kinase C (PKC) inhibitor, GF109203X (1 microM), but not by another PKC inhibitor, H7 (50 microM), whereas TPA-induced MEK1/2-ERK activation was partially and completely inhibited by GF109203X (1 microM) and H7 (50 microM), respectively. A phosphoinositol 3-kinase inhibitor, LY294002, did not inhibit the TPA-induced SEK1-JNK activation. Taken together, these results suggest that activated Ki-Ras-mediated signals are involved in the SEK1-JNK pathway through a PKC isotype that is distinct from that involved in MEK1/2-ERK activation in human colon cancer cells and independent of phosphoinositol 3-kinase activation, and the imbalance between ERK and JNK activity caused by activated Ki-Ras may play critical roles in human colorectal tumorigenesis.     

12-O-tetradecanoylphorbol 13-acetate induced differentiation in human lung squamous carcinoma cells.

Three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) demonstrate features of squamous differentiation including involucrin synthesis and competence to form cornified envelopes. 12-O-Tetradecanoylphorbol 13-acetate inhibits growth of these cell lines and this growth inhibition is associated with enhanced differentiation.     

Suppression of keratinocyte differentiation in SSC-9 human squamous carcinoma cells by benzo[a]pyrene, 12-O-tetradecanoylphorbol-13-acetate and hydroxyurea

In the human squamous carcinoma cell line SCC-9, the expression of two markers of keratinocyte differentiation, involucrin and transglutaminase, was greatly stimulated when growing cultures reached confluence. However, the two markers differed temporally in their induction, with transglutaminase reaching maximal levels shortly after confluence and involucrin a week later. If replication was arrested with hydroxyurea prior to confluence, transglutaminase induction occurred within several days but involucrin levels were completely suppressed. Such a striking degree of uncoupling also resulted when the cells were treated with polycyclic aromatic hydrocarbons such as benzo[a]pyrene but not with 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, or with pyrene. Chronic treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed expression of both transglutaminase and involucrin. However, suppression of the latter (evident in greatly reduced mRNA levels) was considerably more potent and powerful. These findings demonstrate uncoupling of keratinocyte differentiation, potentially useful in analysis of its multiple regulatory influences. They also emphasize the utility of sensitive keratinocyte targets for studying the mechanisms by which model carcinogens disturb the orderly progression of events in their differentiation program.     

Effects of nickel sulfate on growth and differentiation of normal human bronchial epithelial cells

Epidemiological studies have shown that inhalation of nickelcompounds enhances the risk of human respiratory cancer. Culturesof normal human bronchial epithelial cells were continuouslyexposed to a dose (5–20 µg/ml) of NiSO₄ that reducedtheir colony forming efficiency by 30–80%. After 40 daysof incubation, the cultures consisted of large, squamous cells;mitotic cells were not observed. The cells were then maintainedin medium without NiSO₄. After 40–75 total days of incubation,colonies of mitotic cells appeared at a rate of 1 colony per100 000 cells originally at risk; no colonies appeared in controlcultures or in cultures exposed to <5 µg NiSO₄/ml for90 days. Twelve NiSO₄-altered cell cultures isolated from fiveexperiments have been expanded into mass cultures. Most of thecell lines have an increased population doubling potential (>50divisions). Some exhibit aberrations in the squamous (terminal)differentiation process whereas others have lost the requirementfor epidermal growth factor for clonal growth. Aneuploidy andmarker chromosomes have also been noted. However, none of theseNiSO₄-altered cell cultures are anchorage independent nor dothey produce tumors upon injection into athymic nude mice.     

Pathobiological effects of acrolein in cultured human bronchial epithelial cells

The ability of the highly reactive aldehyde acrolein to affect growth, membrane integrity, differentiation, and thiol status and to cause DNA damage has been studied at serum- and thiol-free conditions using cultured human bronchial epithelial cells. Acrolein markedly decreases colony survival at 3 microM whereas about 10-fold higher concentrations are required to increase membrane permeability, measured as uptake of trypan blue dye. Acrolein at micromolar concentrations also causes epithelial cells to undergo squamous differentiation as indicated by decreased clonal growth rate, dose-dependent increased formation of cross-linked envelopes, and increased cell planar surface area. Acrolein causes a marked and dose-dependent cellular depletion of total and specific free low-molecular-weight thiols as well as protein thiols. Exposure to acrolein did not cause oxidation of glutathione indicating that thiol depletion occurred by direct conjugation of reduced glutathione to acrolein without concomitant generation of active oxygen species. Furthermore, acrolein is genotoxic and causes both DNA single strand breaks and DNA protein cross-links in human bronchial epithelial cells. The results indicate that acrolein causes several cytopathic effects that relate to multistage carcinogenesis in the human bronchial epithelium.     

Novobiocin- and phorbol-12-myristate-13-acetate-induced differentiation of human leukemia cells associated with a reduction in topoisomerase II activity

Studies were conducted to determine the possible involvement of DNA topoisomerase II (Topo II) in the induction of differentiation in two human promyelocytic HL-60 leukemia cell variants that are either susceptible or resistant to differentiation induced by phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator. The acquisition of maturation markers and changes in the activity, level, and phosphorylation of Topo II were determined after treatment with either novobiocin, a Topo II inhibitor, or PMA. Novobiocin at 50-150 microM induced differentiation in the HL-205 cells but not in the HL-525 cells, although both cell types were equally susceptible to novobiocin-evoked cytotoxicity. In both cell types, novobiocin induced similar reductions in topoisomerase I activity but different reductions in Topo II activity. Treatment with novobiocin at 150 microM for 6 h or at 2 mM for 30 min resulted in a 4-fold or higher reduction in Topo II activity in the differentiation-susceptible HL-205 cells but not in the differentiation-resistant HL-525 cells. A differential response in Topo II activity was also observed after treatment with PMA. The novobiocin-evoked decrease in Topo II activity seems to be due to an enhanced enzyme proteolysis, whereas the PMA-elicited decrease in Topo II activity is associated with an increase in Topo II phosphorylation. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine, which is an inhibitor of protein kinases, including protein kinase C, diminished the novobiocin-elicited proteolysis of Topo II and the PMA-induced Topo II phosphorylation, as well as the decrease in Topo II activity and the acquisition of differentiation markers induced by either novobiocin or PMA. These results suggest that induction of differentiation in HL-60 cells by novobiocin or PMA is associated with a reduction in Topo II activity, mediated directly or indirectly by a protein kinase(s), perhaps protein kinase C.     

Effects of palytoxin or ouabain on growth and squamous differentiation of human bronchial epithelial cells in vitro

The effects of the non-12-O-tetradecanoylphorbol-13-acetatetype tumor promoter palytoxin on human bronchial epithelialcells was studied in an in vitro serum-free culture system.Unlike the results of previous studies with another tumor promoter,12-O-tetradecanoylphorbol-13-acetate, palytoxin did not inducesquamous differentiation of normal bronchial epithelial cellsand was equally cytotoxic for normal human bronchial epithelialcells, a human lung tumor cell line, and human bronchial epithelialcells immortalized by infection with adenovirus 12-SV40 hybridvirus (BEAS-2B cells). Palytoxin did not induce a change infree cytosolic Ca²⁺ concentration of BEAS-2B cells. The effectof palytoxin on the c-myc mRNA steady state level in BEAS-2Bcells was studied: 1 pM palytoxin increased the steady-statelevel at 12 and 18 h. Furthermore, the induction was accompaniedby an increase in [³H]thymidine uptake. Because palytoxin bindsto (Nat⁺ + K⁺ATPase, the effects of ouabain were compared tothe effects of palytoxin. A ouabain-resistant cell line wasas sensitive to the growth inhibitory effect of palytoxin asthe parent ouabain-sensitive cell line, suggesting differentbinding sites to the (Na⁺ + K⁺-ATPase for palytoxin and ouabain.Ouabain also increased the steady-state level of c-myc geneexpression, but earlier than palytoxin, and the increase inthe level of c-myc mRNA was accompanied by a drop in DNA synthesis.These results suggest that palytoxin does not act by growthstimulation, differential cytotoxicity or terminal differentiationof normal versus neoplastic cells which are proposed mechanismsof tumor promotion.     

The toxic response of preneoplastic rat tracheal epithelial cells to 12-O-tetradecanoylphorbol-13-acetate

The purpose of our studies was to examine the effect of 12-O-tetradecanoylphorbol-13-acetate(TPA) on survival, proliferation, and differentiation of normaland preneoplastic rat tracheal epithelial (RTE) cells. NormalRTE cells obtained from 8-week-old rats were plated into mediumcontaining TPA at concentrations ranging from 0.1 to 100 ng/ml.A dose-related increase in colony-forming efficiency (CFE) wasobserved at concentrations above 0.1 ng/ml. At 100 ng/ml theCFE was increased 5-fold above that observed in controls. Similarstudies were carried out with immortal, preneoplastic RTE cellsderived from carcinogen-exposed primary cultures. Upon exposureto 10 ng/ml of TPA, 9 out of 11 cell lines showed a marked decreasein CFE ranging from a 70 to a >99% reduction. Two out of11 cell lines showed only a minor reduction in CFE at this TPAconcentration. Teleocidin B was as potent as TPA in causinginhibition of CFE in preneoplastic RTE cells; the potency of4-O-methyl TPA was approximately 1/100 that of TPA and phorbolwas virtually inactive. Further studies suggested that inhibitionof clonal growth of the preneoplastic tracheal cells by TPAwas due to rapid cell death. As early as 2 h after additionof TPA to the cultures, a loss of viable cells from the culturesas well as a reduction in CFE was observed. Morphological studiessupport the interpretation that a large number of cells wasseverely damaged within 2–4 h after TPA exposure. Theformation of cross-linked envelopes (CLE), a measure of keratinocytedifferentiation, was determined in cultures of preneoplasticRTE cells. TPA caused only minor increases in CLE formationindicating that the cells were not dying from TPA-induced terminalkeratinocyte differentiation, but rather from TPA toxicity.Thus, preneoplastic RTE cells respond differently to TPA fromnormal RTE cells and also from normal and transformed keratinocytesof various origins. The role of TPA toxicity in promoting preneoplasticRTE cells to the neoplastic state is being investigated.