12-o-retinoylphorbol-13-acetate and mezerein

We have measured the levels of thioredoxin, thioredoxin reductase and glutaredoxin enzyme activity in mouse skin following topical application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator and tumor promoter. The specific activity of thioredoxin and thioredoxin reductase in extracts from normal epidermis increased by 40 and 50%, respectively, after single or multiple application of TPA. Multiple applications (twice per week for 2 weeks) of TPA increased glutaredoxin activity by >300%. Induction of the proteins lasted several days. Other PKC activators, like 12-O-retinoylphorbol 13-acetate, mezerein, 1-oleoyl-2-acetylglycerol and the calcium ionophore A23187, also induced all the enzyme activities. Phorbol and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate, weak activators of PKC, selectively induced the thioredoxin system only and did not influence glutaredoxin activity. Multiple applications of TPA to tumor initiated (7,12-dimethyl[a]benzanthracene-treated) skin resulted in elevated levels of both the thioredoxin and glutaredoxin systems when examined 6 days after the last phorbol ester treatment. Induction of thioredoxin, thioredoxin reductase and glutaredoxin activities by TPA and calcium ionophores may play a general role in the epigenetic mechanism of tumor promotion via thiol redox control mechanisms.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Calcimycin; Calcium; Carcinogens; Cocarcinogenesis; Diglycerides; Diterpenes; Enzyme Activation; Enzyme Induction; Epidermis; Female; Fluocinolone Acetonide; Gene Expression Regulation; Glutaredoxins; Glutathione; Ionophores; Mice; Oxidation-Reduction; Oxidoreductases; Phorbol Esters; Protein Kinase C; Proteins; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Thioredoxin-Disulfide Reductase; Thioredoxins; Tosylphenylalanyl Chloromethyl Ketone; Tretinoin

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.

Topics: Animals; Carcinogens; Catechin; Diterpenes; DNA; DNA Damage; Female; Hydrogen Peroxide; Inflammation; Mice; Mice, Inbred Strains; Neutrophils; Oxidation-Reduction; Phorbol Esters; Skin; Tamoxifen; Terpenes; Tetradecanoylphorbol Acetate

Susceptibility of hairless inbred S/RV Cri-ba or Bare mice to skin tumor development with suboptimal doses of 7,12-dimethylbenz[a]anthracene (DMBA), DMBA-TPA two stage protocol and two stage promotion using 12-O-tetradecanoylphorbol-13-acetate (TPA) as sub-stage 1 promoter and mezerein (MEZ) or phorbol retinoate acetate (PRA) as substage 2 promoter was determined. A single application of 40 or 20 nmol DMBA induced 4-5 papillomas per mouse 40 weeks after initiation while no tumors appeared after similar treatment with 10 or 4 nmol DMBA. Dose response studies for DMBA initiation revealed that 10 nmol DMBA dose saturated the sites for initiation in the resting epidermis. In two stage promotion experiments, MEZ was found to be a potent stage 2 promoter, while PRA acted as a weak complete promoter.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogenicity Tests; Cocarcinogenesis; Diterpenes; Dose-Response Relationship, Drug; Female; Mice; Mice, Hairless; Papilloma; Phorbol Esters; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate

To facilitate the study of skin tumor promotion, a cell culture model system with characteristics analogous to initiated mouse epidermis was established. Cells of the keratinocyte cell line 308, derived from adult mouse skin initiated with 7,12-dimethylbenz[a]anthracene, display the initiated phenotype, since papillomas are produced when the cells are grafted to the backs of athymic mice. Coculture of a small number of these initiated cells with confluent normal primary keratinocytes resulted in the inhibition of growth of colonies of 308 cells. Addition of fresh keratinocytes weekly was required to sustain the inhibition for 3-4 weeks. Inhibition of 308 cell colonies required culture medium with a calcium concentration of 1.2 mM; normal keratinocytes did not inhibit 308 cells in medium with 0.05 mM calcium. Growth of 308 cells was not inhibited by coculture with confluent fibroblasts or by 1.2 mM calcium medium conditioned by either keratinocytes or fibroblasts. During continuous exposure of the cocultures to tumor promoters, 308 cell colonies became apparent within 2-3 weeks. A limited number of promoters were tested in this model system and 12-O-tetradecanoylphorbol-13-acetate, 12-O-retinoylphorbol-13-acetate, mezerein, and benzoyl peroxide were all active. The number of colonies which developed during promoter exposure in cocultures showed a dose-response curve which differed from the dose-response curve for stimulation of growth of 308 colonies in the absence of normal keratinocytes. Simultaneous treatment with 12-O-tetradecanoylphorbol-13-acetate and known inhibitors of skin tumor promotion, such as retinoic acid, fluocinolone acetonide, and bryostatin 1, blocked colony formation of 308 cells in cocultures but not in cultures with only 308 cells. In this model system, the actions of promoters and inhibitors both appear to be mediated by normal keratinocytes.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Animals, Newborn; Antineoplastic Agents; Benzoyl Peroxide; Carcinogens; Cell Communication; Cell Division; Cell Line; Cells, Cultured; Clone Cells; Diterpenes; Keratinocytes; Mice; Mice, Inbred BALB C; Phorbol Esters; Terpenes; Tetradecanoylphorbol Acetate; Tretinoin

Cultured C3H/10T1/2 cells transfected with the plasmid pdBPV-1 were used as targets, and the frequency of transformed colonies as the endpoint to test the enhancing capacity of four promoters: 12-O-tetradecanoylphorbol-13-acetate (TPA), 4-O-methyl-tetradecanoylphorbol-13-acetate (4-O-methyl-TPA), mezerein and phorbol-12-retinoate-13-acetate (PRA). The frequency of the transfected C3H/10T1/2 cells to form transformed colonies was enhanced in the following order: mezerein greater than PRA greater than TPA greater than 4-O-methyl-TPA. The amount of promoters required to promote a tenfold increase in transformed cells was 0.24, 0.81, 30 and 100 ng/ml mezerein, PRA, TPA and 4-O-methyl-TPA, respectively. A significant promoting effect was obtained by a 3.5-day exposure to mezerein regardless of whether it was added at different time intervals after transfection with BPV-DNA. The examined promoters lacked genotoxic activity, as tested on Chinese hamster ovary cells, using chromatid aberrations and exchanges, frequency of macronuclei, unscheduled DNA synthesis (UDS) and inhibition of UDS as endpoints. The usefulness of BPV-1-induced transformation as a bioassay for detecting chemicals with promoting activities is discussed.

Topics: Animals; Bovine papillomavirus 1; Carcinogens; Cell Transformation, Viral; Chromosome Aberrations; Diterpenes; Mice; Mice, Inbred C3H; Micronucleus Tests; Papillomaviridae; Phorbol Esters; Sister Chromatid Exchange; Terpenes; Tetradecanoylphorbol Acetate

We have shown that the second stage tumor promoters mezerein (MEZ) and phorbol 12-retinoate 13-acetate (PRA) inhibit the gluccocorticoid-induced increase in glycerol phosphate dehydrogenase (GPDH) activity in C6 rat glioma cells with ED 50-values of 3.9 and 2.9 nM, respectively. Phorbol 12-myristate 13-acetate (PMA) was 10-fold less potent. MEZ was likewise more potent than PMA for inhibition of cAMP formation in response to isoproterenol. Binding competition studies using [3H]phorbol 12,13-dibutyrate ([3H]PDBu) yielded apparent Ki-values for MEZ and PRA of 50-70 nM. The large difference between the biological potencies of MEZ and PRA and their affinity for the major phorbol ester receptor suggest they may be acting through a more complicated mechanism in these cells.

Topics: Animals; Caenorhabditis elegans Proteins; Carcinogens; Carrier Proteins; Cell Line; Cyclic AMP; Diterpenes; Glioma; Glycerolphosphate Dehydrogenase; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Rats; Receptors, Drug; Receptors, Immunologic; Terpenes; Tetradecanoylphorbol Acetate

This report shows that generation of hydrogen peroxide (H2O2) by human polymorphonuclear leukocytes (PMNs) activated with tumor promoters of varying potency as first and second stage promoters correlates well with activities of these promoters in vivo. Those tested were 12-O-tetradecanoylphorbol-13-acetate (TPA), a complete promoter, 12-O-retinoylphorbol-13-acetate (RPA), a synthetic TPA derivative almost devoid of first stage activity in some strains of mice, and mezerein (Mez), a potent second stage and much weaker first stage promoter. Mez-stimulated PMNs produced up to four times less H2O2, whereas RPA-stimulated PMNs produced up to 10 times less H2O2 than TPA-activated cells when used at concentrations between 0.5 and 15 nM to activate 7.5-8.5 X 10(4) PMNs/ml. Phorbol, a non-promoter, was totally inactive in this assay. Furthermore, the tumor promoter-activated PMNs caused formation of 5-hydroxymethyl-2'-deoxyuridine (HMdU) and thymidine glycol (dTG) in DNA co-incubated with those cells. The amounts of modified thymidines formed, particularly of HMdU, correlated well with first stage tumor promoting efficacy and with the amount of H2O2 that was generated by promoter activated PMNs. In comparison with TPA, Mez- or RPA-stimulated PMNs induced formation of 25 or 70% less H2O2 and 30 or 75% less HMdU, respectively, under conditions favoring HMdU formation. Thus, formation of either H2O2 by tumor promoter-stimulated phagocytes or HMdU in DNA exposed to those activated cells may serve as a measure of potency as a first stage tumor promoter. Formation of modified bases such as HMdU in DNA might constitute the genetic change imparted by the first stage tumor promoters.

Topics: Diterpenes; DNA; Humans; Hydrogen Peroxide; Kinetics; Neutrophils; Phorbol Esters; Terpenes; Tetradecanoylphorbol Acetate

Alterations in NMRI mouse skin induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in "stage I of tumor promotion" are slowly reversible, and this reversibility has a half-time of 10 to 12 weeks. The tumor response observed in the course of an initiation-promotion experiment in vivo is independent of whether stage I of promotion occurs before or after initiation. Since the time interval between treatment with the promoter, and subsequent initiation can be extended up to at least 6 weeks, an enhancement of initiation because of promoter-induced cellular DNA synthesis seems to be unlikely. This result may be inconsistent with the two-stage model of tumor promotion because it indicates that in skin the existence of initiated cells is not required for the induction of cellular alterations that are essential for the stage of skin tumorigenesis called stage I of promotion.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Transformation, Neoplastic; Diterpenes; Female; Mice; Phorbol Esters; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Time Factors

The complete tumour promoter phorbol, 12-myristate, 13-acetate (PMA) induces terminal differentiation in the majority of normal cultured human and mouse keratinocytes but a subpopulation exists which is resistant to this effect (PMAR). We have compared with PMA the effects of mezerein (Mez) and phorbol, 12-retinoate, 13-acetate (PRA) on the ability of normal and transformed human and mouse keratinocytes to terminally differentiate in an attempt to elucidate why the latter two compounds are inefficient complete tumour promoters but are effective as second-stage promoters when given after PMA in the two-stage promotion regimen. Both PMA and Mez increased cornified envelope formation in a similar way in normal and transformed keratinocyte cultures inducing a 20- to 25-fold increase over the solvent controls in normal keratinocytes but only a 2-fold increase in line SCC-27 (a cell line derived from a human squamous cell carcinoma). However, while quantitative dose response studies of the effect of phorbol esters on colony forming ability revealed a proportion of normal human and mouse keratinocytes which were resistant to PMA, no normal keratinocytes were resistant to Mez or PRA. In contrast, cell lines derived from papillomas and squamous cell carcinomas showed a resistant fraction of similar size with all three compounds. Furthermore, when Mez or PRA were mixed with PMA the survival of line SCC-27 was the same as when the cultures were treated with the compounds individually indicating that the keratinocytes which were resistant to PRA or Mez were also the PMAR subpopulation. A non-tumorigenic subclone of line SCC-12 (clone F.2), previously shown to possess all known properties of transformed keratinocytes except defective terminal differentiation in suspension culture responded to PMA and Mez in a similar way to normal keratinocytes, suggesting that resistance of the PMAR subpopulation to second-stage promoters requires the expression of a defect in the keratinocyte terminal differentiation programme.

Topics: Carcinogens; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Diterpenes; Drug Resistance; Epidermal Cells; Epidermis; Humans; Keratins; Phorbol Esters; Phorbols; Terpenes; Tetradecanoylphorbol Acetate