Induction of cyclooxygenase-2 expression by peroxisome proliferators and non-tetradecanoylphorbol 12,13-myristate-type tumor promoters in immortalized mouse liver cells

Increased expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin synthesis, has been associated with growth regulation and carcinogenesis in several systems. COX-2 is known to be induced by cytokines and the skin tumor promoter 12-tetradecanoylphorbol-13-myristate (TPA). In the present study, we investigated the effects of several non-TPA-type tumor promoters on COX-2 expression in immortalized mouse liver cells. Specifically, we tested peroxisome proliferators (PPs), which are rodent liver tumor promoters that cause gross alterations in cellular lipid metabolism, the rodent liver tumor promoter phenobarbital, and the skin tumor promoters okadaic acid and thapsigargin. The PPs Wy-14643, mono-ethylhexyl phthalate, clofibrate, ciprofibrate ethyl ester, and eicosatetraynoic acid each caused large increases in COX-2 mRNA and protein, with maximal expression seen approximately 10 h after treatment of quiescent cells. COX-2 expression was also induced by thapsigargin, okadaic acid, and calcium ionophore A23187, but not by phenobarbital or the steroid PP dehydroepiandrosterone sulfate. Induction of COX-2 expression generally resulted in increased synthesis of prostaglandin E2 (PGE2). However, the PPs caused little or no increase in PGE2 levels, and they inhibited serum-induced PGE2 synthesis. Unlike non-steroidal anti-inflammatory drugs, the PPs do not directly inhibit cyclooxygenase enzyme activity in vitro. Thus, PPs regulate prostaglandin metabolism via both positive (COX-2 induction) and inhibitory mechanisms. In summary, the strong induction of COX-2 expression by PPs, thapsigargin, and okadaic acid suggests a possible role for COX-2 in the growth regulatory activity of these non-TPA-type tumor promoters.     

Ferric nitrilotriacetate promotes N-diethylnitrosamine-induced renal tumorigenesis in the rat: implications for the involvement of oxidative stress

Ferric nitrilotriacetate (Fe-NTA) is a known complete renal carcinogen. In this study we show that Fe-NTA is a potent inducer of renal ornithine decarboxylase (ODC) activity and DNA synthesis and promoter of N-diethylnitrosamine (DEN)-induced renal tumorigenesis in rat. Fe-NTA induced renal ODC activity several fold as compared with saline-treated rats. Renal DNA synthesis, measured as [3H]thymidine incorporation into DNA, was increased after Fe-NTA treatment. Similar to other known tumor promoters, Fe-NTA also depleted the antioxidant armory of the tissue. It depleted glutathione (GSH) levels to approximately 55% of saline-treated controls. It also led to a dose-dependent decrease in the activities of glutathione reductase and glutathione S-transferase. Similarly, activities of catalase, glutathione peroxidase and glucose 6-phosphate dehydrogenase decreased significantly (45-65%). In contrast, gamma-glutamyl transpeptidase activity showed an increase. The maximum changes in activities of these enzymes could be observed at 12 h following Fe-NTA treatment. In addition, Fe-NTA augmented renal microsomal lipid peroxidation >150% over saline-treated controls, which was concomitant with the alterations in GSH metabolizing enzymes and depletion of the antioxidant armory. These effects were alleviated in rats which received a pretreatment with an antioxidant, BHA or BHT. Fe-NTA promoted DEN-induced renal tumorigenesis. In saline alone- and DEN alone-treated animals no tumors could be recorded, whereas in Fe-NTA alone-treated animals 17% tumor incidence was observed. However, in DEN-initiated and Fe-NTA-promoted animals tumor incidence increased to 71%. Our results show that Fe-NTA induces oxidative stress in the kidney and decreases antioxidant defenses, as indicated by the fall in GSH level and in the activities of glutathione peroxidase and catalase. Concomitantly, Fe-NTA increases ODC activity and DNA synthesis, which may be compensatory changes following oxidative injury to renal cells in addition to providing a strong stimulus for renal tumor promotion. Thus oxidative stress and impaired antioxidant defenses induced by Fe-NTA in the kidney may contribute to the observed nephrotoxicity and carcinogenicity.     

Activation of the epidermal growth factor receptor by skin tumor promoters and in skin tumors from SENCAR mice

The present study was designed to further investigate the role of the epidermal growth factor receptor (EGFr) in mouse skin tumor promotion by evaluating the status of the EGFr in tumor promoter-treated mouse epidermis and in mouse skin tumors. Female SENCAR mice received three topical treatments of either the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or the nonphorbol esters okadaic acid and chrysarobin. Membrane proteins from SENCAR mouse epidermis were isolated 6 h after the last treatment, and the phosphotyrosine content of the EGFr and several potential substrates were examined by Western blot analysis. The results indicated that multiple applications of all three tumor promoters led to an increase in the phosphotyrosine content of the EGFr and also of several lower molecular weight proteins (M(r) approximately 80,000-85,000). Phosphorylation of PLC gamma 1 on tyrosine residues could not be detected in tumor promoter-treated mouse epidermis when the phosphotyrosine content of the EGFr was elevated or in cultured keratinocytes exposed to exogenous EGF. When two tyrosine kinase inhibitors (tyrphostins RG50864 and RG13022) were incorporated into the treatment regimens, the TPA-induced epidermal hyperplasia and cell proliferation were effectively blocked, and the TPA-stimulated EGFr tyrosine phosphorylation was significantly reduced. Examination of the phosphotyrosine content of epidermal membrane proteins isolated from skin papillomas revealed that the EGFr also had elevated phosphotyrosine levels. These results demonstrate that multiple topical treatments with both phorbol ester and nonphorbol ester tumor promoters lead to activation of the EGFr tyrosine kinase in mouse epidermis. In addition, these data suggest that signaling through the EGFr pathway plays an important role in the tumor promotion stage of multistage carcinogenesis in mouse skin.     

Activation of erbB2 and c-src in phorbol ester-treated mouse epidermis: possible role in mouse skin tumor promotion

In recent work we showed that the EGF receptor (EGFr) was activated in tumor promoter treated mouse epidermis (Cell Growth & Differentiation, 6: 1447-1455, 1995). In the present study, we have investigated the possible role of other erbB family members in the process of tumor promotion. Both erbB2 and erbB3, but not erbB4, were expressed in cultured mouse keratinocytes and in mouse epidermis in vivo. In cultured mouse keratinocytes, EGF stimulated rapid tyrosine phosphorylation of erbB2 followed by a time-dependent degradation of erbB2 protein. Furthermore, an increase in erbB2:EGFr heterodimer formation was also induced by EGF. In contrast to the results with erbB2, EGF did not induce tyrosine phosphorylation, the degradation of erbB3, or erbB3:EGFr heterodimer formation in cultured keratinocytes. Further analyses revealed that c-src kinase activity was dramatically elevated in cultured mouse keratinocytes exposed to EGF. In mouse epidermis following multiple treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA), the phosphotyrosine content of erbB2 was significantly elevated in a dose-dependent manner. Concomittantly, erbB2:EGFr heterodimer formation and c-src kinase activity were also elevated in TPA-treated epidermis. Structure-activity relationships with several phorbol ester analogs showed that the elevated phosphorylation of erbB2 in mouse epidermis followed closely with tumor promoting ability. Activation of erbB2 and c-src kinase were also observed in the epidermis of TGF alpha transgenic mice where expression of human TGF alpha was targeted to basal keratinocytes with the human K14 promoter. Collectively, the current data suggest that the activation of erbB2 in phorbol ester treated skin can be explained solely by a mechanism involving elevation of EGFr ligands and activation of the EGFr. In addition, activation of c-src may be an important downstream effector in mouse keratinocytes both in vivo and in vitro, following activation of the EGFr, erbB2, or both.     

Effect of a chemical carcinogen and phorbol esters on sterol metabolism of mouse skin

The effect of 20-methylcholanthrene and phorbol esters on sterol metabolism of mouse skin was studied. When 4 beta-phorbol esters were administered to mice that were previously painted once with 20-methylcholanthrene, a depression of some sterols in skin occurred, of which that of lathosterol was most marked. This effect was not observed when the order of application was reversed. Using a metabolic inhibitor, diazacholesterol, it was shown that sterols which reduce in mouse skin by administration of carcinogen and promoters were similar to those which reduce by administration of carcinogen only and are the members of one of the two cholesterol-biosynthetic pathways, i.e., a pathway which proceeds through intermediates with a saturated side chain. The intensity of the lathosterol-depressing effect of phorbol esters depends on the order of application of 20-methylcholanthrene and promoters, the amount of promoters, molecular species of alcoholic moiety of esters, and configuration at C-4 of phorbol moiety. Of the phorbol esters tested, 4 beta-phorbol-12-myristate-13-acetate revealed the highest activity, which was followed by 4 beta-phorbol-12,13-didecanoate, 4 beta-phorbol-12,13-dibutyrate, 4 beta-phorbol-12,13-dibenzoate, 4 beta-phorbol-12,13-diacetate, 4 alpha-phorbol-12,13-didecanoate, and 4 alpha-phorbol. 4 alpha-Phorbol was practically inactive. When beta-naphthoflavone was substituted for 20-methylcholanthrene, little effect was observed except in TPA, which revealed a rather marked lathosterol-depressing activity. Phorbol esters themselves did show some activity of lathosterol depression without prior application of 20-methylcholanthrene, but the effects were much weaker. When anthralin was applied to mouse skin after the painting of 20-methylcholanthrene, a low but definite lathosterol-depressing effect was observed.     

Analysis of the ability of 12-O-tetradecanoylphorbol-13-acetate to induce epidermal hyperplasia, transforming growth factor-alpha, and skin tumor promotion in wa-1 mice

Wa-1 mutant mice possess a defect in the production of transforming growth factor-alpha (TGF-alpha) that leads to a phenotype characterized by wavy hair and curly whiskers. In light of recent evidence indicating the importance of TGF-alpha in epithelial tumorigenesis, this study characterizes the responsiveness of wa-1 mice to skin tumor promotion by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). The responsiveness of wa-1 mice to TPA was compared with that of SENCAR and C57BL/6 mice, representing mouse lines highly sensitive and resistant to skin tumor promotion, respectively. Wa-1 mice were found to be very resistant to skin tumor promotion by TPA after initiation with 10 nmol DMBA, similar to C57BL/6 mice. TPA failed to induce a dramatic increase in TGF-alpha mRNA and protein in the skin of wa-1 mice, whereas TGF-alpha mRNA and protein were dramatically induced in the skin (both epidermis and dermis) of SENCAR and C57BL/6 mice. TPA treatment dramatically increased mRNA levels of two other EGF receptor ligands, amphiregulin and heparin binding-EGF, however, in the skin of all three mouse lines. Comparison of histologic changes in skin revealed that wa-1 mice exhibited only modest sustained epidermal hyperplasia after multiple treatments with TPA, similar in magnitude to that of C57BL/6 mice and significantly lower than that of SENCAR mice. The current data indicate that wa-1 mice are relatively resistant to TPA promotion. Possible mechanisms for this resistance are discussed.     

Inhibition of the tumor promoting action of 12-O-tetradecanoylphorbol-13-acetate by tubeimoside III isolated from Bolbostemma paniculatum

As tubeimoside I isolated from Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae) has been shown to suppress tumor promoter effects, tubeimoside III from the same plant was tested in vitro and in vivo against the action of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Tubeimoside III, the natural analog of tubeimoside I, also had an anti-inflammatory effect on mouse ear edema induced by arachidonic acid and TPA and a potent anti-tumor promoting effect on two-stage carcinogenesis of mouse skin after topical application. However, the important difference in bioactivities between tubeimosides I and III is the non-activity of tubeimoside III as an inhibitor of tumor promotion if administered orally. Differences in metabolism connected with different routes of the compound may be one of a number of explanations of the important difference.     

Growth inhibition of human melanoma-derived cells by 12-O-tetradecanoyl phorbol 13-acetate

In vitro growth of 6 human melanoma-derived cell lines was inhibited markedly by the phorbol-ester tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA), a potent activator of several isoforms of protein kinase C (PKC). Utilizing PKC isoform-specific antibodies in immunoblotting experiments, we found that the PKC alpha and PKC epsilon isoforms were expressed in all of the 6 melanoma cell lines tested, whereas the PKC beta isoform was expressed at detectable levels in only 2 of the 6 cell lines. The SK-Mel-173 melanoma cell line, which had relatively high levels of PKC beta mRNA and protein expression, and which was also the most sensitive to cell growth inhibition by TPA, was used to isolate clones whose growth was less inhibited by TPA. Immunoblotting experiments revealed that in parental SK-Mel 173 cells PKC beta was rapidly down-regulated to below detectable levels after treatment for 48 hr with TPA, but that in TPA-resistant variant clones there was negligible down-regulation of PKC beta by TPA. On the other hand, treatment of parental and TPA-resistant SK-Mel 173 cells with TPA led to partial down-regulation of PKC alpha in both cell lines. Total PKC enzyme activity was also greater in TPA-resistant cells than in parental SK-Mel 173 cells. Our results show that TPA might inhibit the growth of melanoma cells by causing down-regulation of specific isoforms of PKC that are required to maintain the growth of these cells.     

Anti-tumor-promoting activity of majonoside-R2 from Vietnamese ginseng, Panax vietnamensis Ha et Grushv. (I)

Seven saponins (1-7) isolated from the rhizomes and roots of Panax vietnamensis were tested for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), in Raji cells as a primary screening test for anti-tumor-promoters (cancer chemopreventive agents). The ocotillol-type saponin, majonoside-R2 (2), which is the major and characteristic constituent of this plant, exhibited a significant inhibitory effect on EBV-EA activation. Furthermore, the cell cycle analysis of 2 on Raji cells was also examined and strong inhibition was observed on the effect of the cell cycle induced by TPA. Compound 2 showed potent anti-tumor-promoting activity in two-stage carcinogenesis tests of mouse skin using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA or fumonisin B1 as a promoter. Consequently, these results suggest that majonoside-R2 (2) could be a valuable chemopreventive agent against chemical carcinogenesis.     

Transcriptional regulation of prostaglandin-H synthase-2 gene in human trophoblasts

Abnormal PG production by placental PG-H synthase (PGHS) is associated with preeclampsia. There are two PGHS isozymes, and their regulation in trophoblasts is presently unknown. We hypothesized that the PGHS isozymes are differentially regulated in human trophoblasts. To test this hypothesis, we transfected primary trophoblasts and JEG3 cells with promoter constructs of either PGHS-1 or PGHS-2 genes. We found that in both cell systems, the basal activity of PGHS-2 promoter was 10- to 30-fold higher than the activity of PGHS-1 promoter. In response to either 12-0-tetradecanoylphorbol-13-acetate (TPA) or 8-bromo-cAMP, we observed an increase in PGHS-2 promoter activity but no change in activity of PGHS-1 promoter. Similarly, both agents enhanced PGHS-2 expression, as well as prostaglandin E2 production. The activity of PGHS-2 promoter was potentiated by coexpression of protein kinase A and inhibited by coexpression of kinase A inhibitor. Aspirin attenuated the stimulatory effect of TPA on PGHS-2 promoter. We conclude that both PGHS-1 and PGHS-2 promoters are active in trophoblasts. The activity of PGHS-2 promoter is stimulated by either TPA or cAMP, and the stimulatory effect of TPA is attenuated by aspirin. These pathways may play a role in modulation of prostanoid synthesis by trophoblasts.     

Differential selectivity of ligands for the C1a and C1b phorbol ester binding domains of protein kinase Cdelta: possible correlation with tumor-promoting activity

Protein kinase C (PKC) represents the major, high-affinity receptor for the phorbol esters as well as for a series of structurally diverse natural products. The phorbol esters function by binding to the tandem C1a and C1b domains in PKC, leading to enzyme activation. Although the typical phorbol esters represent the paradigm for tumor promoters in mouse skin, it is now clear that different high affinity ligands for PKC have distinct biological effects. Thus, the daphnane analogue mezerein is a second-stage promoter, the macrolide bryostatin 1 is a partial antagonist, and certain 12-deoxyphorbol 13-monoesters also function as partial antagonists but with a different pattern of activity. The biochemical basis for these differences is an area of active investigation. In this report, we have examined the relative interaction of ligands differing in structure and pattern of biological response with the C1a and C1b domains of PKCdelta. We mutated either or both of the C1 domains of PKCdelta, expressed the constructs in NIH 3T3 cells, and monitored the interaction of the ligands by their ability to induce translocation of the mutated PKCdelta from the cytosol to the particulate fraction. We found that different ligands showed different dependence on the C1a and C1b domains for translocation. Whereas phorbol 12-myristate 13-acetate and the indole alkaloids indolactam and octylindolactam were selectively dependent on the C1b domain, selectivity was not observed for mezerein, for the 12-deoxyphorbol 13-monoesters prostratin or 12-deoxyphorbol 13-phenylacetate, or for the macrocyclic lactone bryostatin 1. Provocatively, the pattern of response corresponds with the activity of the compounds as complete tumor promoters.     

Relationship of oxidative events and DNA oxidation in SENCAR mice to in vivo promoting activity of phorbol ester-type tumor promoters

Reactive oxygen species (ROS) have been implicated as being involved in tumor promotion processes. However, the mechanism by which ROS modulate tumor promotion has not as yet been elucidated. In this report, we show that phorbol ester-type tumor promoters (12-O-tetradecanoylphorbol-13-acetate [TPA], mezerein and 12-O-retinoylphorbol-13-acetate [RPA]), which vary in their in vivo potencies, also differ in their effect on formation of hydrogen peroxide (H2O2) and oxidation of normal bases to 5-hydroxymethyl-2'-deoxyuridine [HMdU] and 8-hydroxyl-2'-deoxyguanosine [8-OHdG] in the DNA of SENCAR mouse epidermis, though they are equipotent in causing infiltration of polymorphonuclear leukocytes (PMNs). Treatment of SENCAR mice with the chemopreventive agents (-)-epigallocatechin gallate or tamoxifen (6.5 nmol) prior to application of TPA (6.5 nmol) diminished PMN infiltration, and formation of H2O2, HMdU and 8-OHdG. These results strengthen the evidence that ROS are involved in tumor promotion, and that generation of ROS and the subsequent oxidative DNA modification are related to the tumor-promoting potencies of the different phorbol ester-type promoters.     

Excimer laser angioplasty

Increased expression of interleukin-1 (IL-1) in skin elicits a variety of responses, including inflammation and epidermal hyperplasia, which are also characteristic events elicited by tumor promoters. The goal of this study was to investigate whether various classes of tumor promoters increase expression of IL-1 alpha and whether phorbol ester-induced IL-1 alpha expression can be blocked by antitumor promoters. Northern analysis of mRNA isolated from the dorsal skins of SENCAR mice treated with 1 microgram of 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA) showed that a single application of TPA produced a significant increase in IL-1 alpha mRNA at 6 h that decreased by 24 h after treatment. Two treatments of TPA at 48-h intervals induced, at 6 h, twice as much IL-1 alpha mRNA as one treatment. Of the other promoters tested, anthralin (22.6 micrograms), mezerein (2 micrograms), calcium ionophore A23187 (120 micrograms), and benzoyl peroxide (20 mg) induced IL-1 alpha mRNA with different kinetics and to different extents. On the other hand, the non-tumor promoting phorbol ester analogue 4 alpha-12-O-tetradecanoylphorbol-13-acetate had little effect on the expression of IL-1 alpha mRNA. The effects of various antitumor promoters on TPA-induced IL-1 alpha mRNA expression were also assessed. Fluocinolone acetonide, mepacrine, and 5,8,11,14-eicosatetraynoic acid were the most effective inhibitors, and each produced about 80% inhibition. Other antitumor promoters such as retinoic acid, N-tosyl-L-phenylalanine chloromethyl ketone, and butylated hydroxytoluene inhibited approximately 35%, 65%, and 50% of TPA-induced IL-1 alpha mRNA expression, respectively. Therefore, this study suggests a possible role of IL-1 alpha in the promotion stage of skin carcinogenesis.     

Clarification of the binding mode of teleocidin and benzolactams to the Cys2 domain of protein kinase Cdelta by synthesis of hydrophobically modified, teleocidin-mimicking benzolactams and computational docking simulation

Phorbol esters (12-O-tetradecanoylphorbol 13-acetate; TPA) and teleocidins are known to be potent tumor promoters and to activate protein kinase C (PKC) by binding competitively to the enzyme. The relationship between the chemical structures and the activities of these compounds has attracted much attention because of the marked structural dissimilarities. The benzolactam 5, with an eight-membered lactam ring and benzene ring instead of the nine-membered lactam ring and indole ring of teleocidins, reproduces the active ring conformation and biological activities of teleocidins. Herein we describe the synthesis of benzolactams with hydrophobic substituents at various positions. Structure-activity data indicate that the existence of a hydrophobic region between C-2 and C-9 and the steric factor at C-8 play critical roles in the appearance of biological activities. We also computationally simulated the docking of teleocidin and the modified benzolactam molecules to the Cys2 domain structure observed in the crystalline complex of PKCdelta with phorbol 13-acetate. Teleocidin and benzolactams fitted well into the same cavity as phorbol 13-acetate. Of the three functional groups hydrogen-bonding to the protein, two hydrogen-bonded with protein atoms in common with phorbol 13-acetate, but the third one hydrogen-bonded with a different protein atom from that in the case of phorbol 13-acetate. The model explains well the remarkable difference in activity between 5 and its analogue having a bulky substituent at C-8.     

Activities of conjugating and antioxidant enzymes following endotoxin exposure

Endotoxin exposure elicits various responses in mammals including the acute phase response that has been shown to cause changes in the activity of several forms of cytochrome P450s and other enzymes. Therefore, the hepatic conjugating enzyme, glutathione S-transferase (GST), and UDP-glucuronosyltransferase (UDPGT), the antioxidant enzymes, glutathione peroxidase (GSHPx), catalase, and superoxide dismutase (SOD), as well as lipid peroxidation were investigated following the administration of endotoxin to male Sprague-Dawley rats (8 mg/kg body weight). Rats were euthanized at various times following endotoxin administration and the livers removed and processed to assess various enzyme activities. Glutathione S-transferase, UDPGT, and GSHPx activity showed statistically significant decreases after 24 hours and remained lower than controls for the duration of the study. Decreases in total SOD and catalase activities were seen at 24, 48, and 72 hours following endotoxin administration; however, only catalase activity showed statistically significant differences between control and treated samples at those time points, and total SOD activity showed a statistically significant decrease at 24 hours. No statistically significant changes were seen in the level of lipid peroxidation in the liver microsomes from endotoxin-treated animals. Changes in the conjugative enzymes and the free-radical scavenging enzymes following endotoxin exposure may alter the host's metabolism and response to free radicals.     

Focal transgene expression associated with papilloma development in v-Ha-ras-transgenic TG.AC mice

The homozygous transgenic mouse line TG.AC contains a v-Ha-ras transgene and rapidly develops epidermal papillomas in response to either wounding or treatment with tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). The transgenic v-Ha-ras protein product was detected in all papillomas removed from TPA-treated TG.AC mice but not in vehicle- or TPA-treated TG.AC skin without tumors. In situ hybridization demonstrated that focal expression of the transgene was limited to regions of papilloma development and further localized the expression of the transgene message to the epidermal component of the papillomas, with the strongest signal in the basal epidermoid cells. Cellular proliferation, as indicated by immunohistochemical staining for proliferating-cell nuclear antigen (PCNA), was similarly localized primarily to basal epidermoid cells and, to a lesser extent, stratum spinosum cells in all papillomas analyzed. Cells that stained positively for PCNA were much more common in the papillomas than in the surrounding, normal-appearing skin. The focal nature of papilloma development was also evidenced by protein kinase C activity and hyperplasia after TPA treatment. As early as 18 d after the start of TPA treatment, focal hyperplasias associated with the follicular epidermis were observed in TG.AC but not nontransgenic FVB/N skin; these hyperplasias were assumed to be the precursors of the epidermal papillomas. To explain the development of transgene-expressing tumors from apparently transgene-negative, normal-appearing skin, we hypothesize that the papillomas arise from the clonal expansion of focal areas of epidermal cells that overexpress the transgene. We also propose that the TG.AC line is an excellent model for studying very early events in papillomagenesis.     

Progesterone stimulates the activity of the promoters of peripheral myelin protein-22 and protein zero genes in Schwann cells

To understand better the mechanisms by which progesterone (PROG) promotes myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (P0) genes. PROG stimulated the P0 promoter and promoter 1, but not promoter 2, of PMP22. The effect of PROG was specific, as estradiol and testosterone only weakly activated promoters. Dose-response curves for stimulation of both promoter constructs by PROG were biphasic. RU486, a PROG antagonist, did not abolish the effect of PROG, but stimulated promoter activities by itself. In the human carcinoma cell line T47D expressing high levels of PROG receptor, PROG did not stimulate the P0 and PMP22 promoters, whereas the promoter region of the mouse mammary tumor virus was fully activated. Thus, the activation by PROG of promoter activity of two peripheral myelin protein genes is Schwann-cell specific.     

Epidermal cell proliferation and promoting ability of phorbol esters

Dose-response relationships on the abilities of several phorbol ester tumor promoters to promote skin tumors after 7,12-dimethylbenz[a]anthracene initiation and to bring about edema, inflammation, and epidermal hyperplasia were determined in female Charles River CD-1 mice. The promoting ability of the potent synthetic promoter, phorbol-12,13-dioctanoate (PdiC8), was determined over a dose range of 0.1-10 mug/application. Administration of PdiC8 two times weekly at dosages of 4, 6, 8, and 10 mug gave little variation in tumor response. A dose-dependent tumor response occurred at doses of 1-4 mug PdiC8. Only 1 papilloma was observed when PdiC8 was given twice weekly at a dose of 0.1 or 0.5 mug. A similar dose-response relation was observed for the ability of PdiC8 to stimulate epidermal hyperplasia. Investigations of other phorbol esters revealed an excellent correlation between their promoting ability and their ability to induce epidermal hyperplasia; however, that was not the case for compounds outside the phorbol ester series (i.e., acetic acid, cantharidin, and ethylphenylpropiolate).