12-o-retinoylphorbol-13-acetate and Skin-Neoplasms

Topics: Animals; Carcinogens; Cell Transformation, Neoplastic; Mice; Models, Biological; Papilloma; Phorbol Esters; Precancerous Conditions; Skin Neoplasms; Tetradecanoylphorbol Acetate

By introduction of conjugated double bonds into the long-chain fatty acid residue of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) the promoting efficacy is abolished, whereas hyperplasiogenic and irritant activities are not impaired. By means of such "disarmed" phorbol esters (with 12-O-retinoylphorbol-13-acetate, RPA, being the most suitable one) the process of skin tumor promotion can be divided into two stages. Stage 1, brought about by short-term (single) treatment with TPA, leads via an induction of cellular proliferation to an apparently irreversible change in skin which is proposed to involve the expression of the neoplastic phenotype. A subsequent long-term (multiple) treatment with RPA (stage 2), results in the appearance of papillomas. There is no positive evidence for a critical role of phorbol ester receptor occupancy and protein kinase C activation or of free radicals such as superoxide anions in stage 1. Retinoic acid inhibits stage 1 only when applied several hours prior to TPA, whereas indomethacin exhibits the strongest inhibitory effect on stage 1 when applied 3 hr after TPA. The indomethacin effect can be specifically overcome by prostaglandin F2 alpha (PGF2 alpha) and correlates with an accumulation of PGF2 alpha in skin 3-4 hr after TPA treatment. After 12-O-retinoylphorbol-13-acetate (RPA) application no prostaglandin accumulation is seen at this time. It is proposed that the expression of the neoplastic phenotype ("conversion" of initiated cells) is accomplished in the course of a PGF2 alpha-mediated metaplastic process which normally plays a physiological role in the wound response.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Caenorhabditis elegans Proteins; Carrier Proteins; Cell Division; Humans; Mice; Phorbol Esters; Phorbols; Protein Kinase C; Receptors, Drug; Receptors, Immunologic; Skin; Skin Neoplasms; Structure-Activity Relationship; Tetradecanoylphorbol Acetate

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

The role of transforming growth factor-beta 1 (TGF-beta 1) in multisage carcinogenesis in mouse skin was assessed by studying its growth inhibitory effects on nontumorigenic and tumorigenic keratinocytes and by examining its mRNA expression in vitro and during epidermal hyperproliferation and multistage carcinogenesis. While growth of primary basal keratinocytes was inhibited by TGF-beta 1 in doses as low as 0.1 ng/mL, the immortal keratinocyte line MCA/3D ("putatively initiated" cells) responded to TGF-beta 1 with slightly reduced sensitivity, and the papilloma-producing keratinocyte line 308 was considerably less sensitive. In contrast, the squamous carcinoma cell line Carc B was completely nonresponsive, and two other tumorigenic cell lines (PDV and PDVC57) were sensitive to growth inhibition by TGF-beta 1. Steady-state levels of TGF-beta 1 mRNA were high in all the malignant cell lines and in line 308 papilloma cells, but low in primary basal cells and in the nontumorigenic keratinocyte lines V2, Reb1, and MCA/3D. Our in vivo studies showed that tumor promoters, but not mitogenic or weak hyperplasiogenic agents, were able to induce transient expression of TGF-beta 1 mRNA in mouse epidermis. A constitutive overexpression of TGF-beta 1 mRNA was observed in malignant carcinomas but not in the benign premalignant lesions, indicating that overexpression may be associated with malignant progression.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Animals, Newborn; Blotting, Northern; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; DNA; DNA Replication; DNA, Neoplasm; Female; Gene Expression; Keratinocytes; Mice; Mice, Inbred Strains; Phorbol Esters; RNA; RNA, Messenger; RNA, Neoplasm; Skin; Skin Neoplasms; Skin Physiological Phenomena; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

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

Using the multistage (initiation-conversion-promotion) protocol we have studied the effects of methyl methanesulfonate (MMS), a well-known alkylating and clastogenic agent, on tumor development in NMRI mouse skin in vivo. When topically applied in a dose up to 400 mumol MMS did not exhibit any initiating efficacy while under identical conditions chromosomal damage (mainly breaks and gaps) was induced in epidermis. A dose of 100 mumol MMS was found to be almost as clastogenic as 10 nmol of the convertogenic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), whereas the non-convertogenic promoter 12-O-retinoylphorbol-13-acetate (RPA, 10 nmol) did not induce chromosomal aberrations in vivo. In combination with initiation by 7,12-dimethylbenz[a]anthracene (DMBA) and promotion by RPA, MMS (2 x 100 mumol) turned out to be a rather powerful convertogenic agent ('stage I tumor promoter'). This tumor-inducing efficacy of MMS was synergistically increased by simultaneous application of RPA. These results support the concept that the induction of chromosomal aberrations plays an important role in skin tumor development, i.e. in the conversion stage.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Chromosome Aberrations; Female; Methyl Methanesulfonate; Mice; Phorbol Esters; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate

Mechanical wounding provides a convertogenic ("stage I tumor-promoting") stimulus in initiated NMRI mouse skin, indicating that this stage of carcinogenesis can be entirely controlled by endogenous factors. A search for such factors led to the finding that both platelet-derived Epstein-Barr-virus-inducing factor (EIF), alias human TGF beta 1 and porcine TGF beta, exhibited--upon intracutaneous injection--convertogenic efficacy in initiated NMRI-mouse skin in vivo provided that their injection was combined with a single topical application of the non-convertogenic tumor promoter 12-O-retinolyphorbol-13-acetate (RPA). Since TGF beta inhibits epidermal cell proliferation, the RPA treatment is thought to provide a mitogenic stimulus required for conversion. The RPA treatment can be replaced by intracutaneous injection of transforming growth factor alpha (TGF alpha). These results indicate that the stage of conversion consists of two components, one of which is related to mitogenesis (RPA or TGF alpha), the other to a still unknown activity exhibited by TGF beta-like factors. Thus, endogenous factors with the quality of "wound hormones" may be involved in multistage skin carcinogenesis. This finding could explain the convertogenic effect of skin wounding.

Topics: Animals; Cell Division; Cells, Cultured; Female; Kinetics; Mice; Mice, Inbred Strains; Phorbol Esters; Skin; Skin Diseases; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factors; Wounds and Injuries

When applied to NMRI mouse skin in vivo, phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12-O-retinoylphorbol-13-acetate (RPA) have been found to induce two early waves of prostaglandin E2 (PGE2) synthesis after 15 and 90 min and a delayed accumulation of prostaglandin F2 alpha (PGF2 alpha) after 2 h. With respect to PGF2 alpha formation different activities of both agents were observed, in that TPA but not RPA induced additional PGF2 alpha waves after 4 and 17 h. Functionally, PGE2 was previously shown to be an endogenous mediator of the TPA- or RPA-induced epidermal hyperproliferation and hyperplasia. A functional role of PGF2 alpha could be attributed to the post-initiation stages of tumor development in initiated mouse skin, i.e. the conversion stage (stage I of tumor promotion) elicited by two TPA applications and the promotion stage (stage II of promotion) brought about repetitive RPA treatments. PGF2 alpha, appearing as one distinct biosynthetic wave 3-4 h after TPA application seems to be critically involved in the conversion steps since (i) inhibition of its accumulation by indomethacin led to an inhibition of tumor formation, (ii) the inhibitory effect of indomethacin could be reversed by PGF2 alpha and (iii) RPA was not able to give rise to the accumulation of PGF2 alpha 4 h after application as obtained by TPA treatment. Moreover, RPA-induced promotion of DMBA- and TPA-treated mouse skin was inhibited by indomethacin. The inhibitory effect of indomethacin on papilloma formation was again reversed by PGF2 alpha treatment concomitant with RPA.

Topics: Animals; Cocarcinogenesis; Dinoprost; Dinoprostone; Epidermis; Indomethacin; Mice; Papilloma; Phorbol Esters; Skin Neoplasms; Tetradecanoylphorbol Acetate

The extracellular matrix of the dermis is subject to severe alterations during tumor promotion with phorbol esters in mouse skin. The metabolic changes also involve general stimulation of protein synthesis and most specifically an increase of collagen synthesis. During chronic treatment with the tumor promoting phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12-O-retinoylphorbol-13-acetate (RPA) increased protein synthesis was observed that did not occur during treatment with the non-promoting mitogens 4-O-methyl-TPA and Ca-ionophore A 23187. Relative collagen synthesis measured as the ratio of radioactivities in hydroxyproline and proline or as the proportion of total radioactivity in pepsin resistant material was elevated, too, but not sufficiently to substitute for TPA-induced collagen loss. In contrast collagen degradation caused by the non-promoting irritant A 23187 is followed by an immediate, substantial increase of collagen synthesis. When TPA treatment was discontinued after a few applications insufficient for tumor development rapid resynthesis of collagen took place. Therefore we assume that continued phorbol ester application not only caused connective tissue damage but also prevents the repair of that damage. This effect seems to be promoter specific and contributes to the disruption of dermal-epidermal interactions during tumor promotion.

Topics: Animals; Calcimycin; Collagen; Female; Mice; Phorbol Esters; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate

Chronic treatment with the tumor-promoting phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12-O-retinoylphorbol-13-acetate (RPA) causes permanently increased levels of active collagenolytic enzymes in the dermis and leads to a stable reduction of dermal collagen content. Non-promoting skin mitogens like the Ca-ionophore A 23187 or the 4-O-methylether of TPA, while being active stimulators of collagenolytic enzymes, do not support chronic collagen degradation throughout the experimental period. On the other hand, TPA-induced collagen degradation is not necessarily influenced by inhibition of tumor promotion. Fluocinolone acetonid (FA), an inhibitor preventing not only tumor development but also chronic inflammation and the establishment of a stationary hyperplasia, has been compared with retinoic acid (RA) which has no influence on either the inflammatory reaction or hyperplasia. While FA inhibited the dermal effects of TPA almost completely, RA at a dose that prevented tumor development by 80% had no effect whatsoever in this respect. Therefore, we conclude that both epidermal proliferation and inflammation are accompanied by collagenolytic reactions in the dermis. During chronic treatment sustained collagenolysis correlates with inflammation and/or the establishment of a stationary hyperplasia. Like these it can be regarded as a necessary but insufficient condition of tumor promotion (second stage).

Topics: Animals; Calcimycin; Cell Division; Collagen; Female; Fluocinolone Acetonide; Mice; Phorbol Esters; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin

Recent evidence shows that stage I of tumor promotion in NMRI-mouse skin induced by a single dose of 12-O-tetradecanoylphorbol-13-acetate (TPA) can be effected not only after initiation by 7,12-dimethylbenz[a]anthracene (DMBA) but also several weeks prior to initiation. In view of this partial inversion of the initiation-promotion sequence we proposed to replace the term 'stage I of promotion' by the term 'conversion'. The convertogenic effectivity of a single dose of TPA applied after DMBA can be suppressed in a rather characteristic and non-toxic fashion by hydroxyurea (HU). In the present study we asked whether HU might also interfere with the converting effectivity of a single dose of TPA given prior to DMBA. NMRI mice received a single dose of TPA 3 weeks prior to initiation by DMBA which was followed by twice weekly application of skin irritant 12-O-retinoylphorbol-13-acetate (RPA) in order to effect stage II of promotion. A single dose of HU given i.p. at different times after TPA was found to interfere with tumor formation exhibiting an almost complete inhibition if administered 18 h after TPA. This inhibition did not prevent a subsequent promotion by repetitive TPA treatment. The data indicate that conversion can be inhibited by HU in the same characteristic fashion regardless of whether TPA was administered after or prior to initiation. The data also support the autonomous character of the conversion process for which epidermal DNA synthesis appears to be obligatory.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cocarcinogenesis; DNA; Female; Hydroxyurea; Mice; Mice, Inbred Strains; Phorbol Esters; Phorbols; Skin Neoplasms; 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 semi-synthetic phorbol ester 12-O-retinoyl phorbol 13-acetate (RPA) was tested for activity as a complete tumor promoter, as well as for first or second stage promotion activity, in the skin of SENCAR mice. Unlike in NMRI mice where RPA has been reported to act only as a second stage promoter, in SENCAR mice it has moderate complete promoting activity as well as second stage activity. RPA also induces epidermal hyperplasia and an increase in the number of dark basal keratinocytes.

Topics: Animals; Female; Mice; Mice, Inbred Strains; Phorbol Esters; Phorbols; Skin Neoplasms; Species Specificity

In order to evaluate the significance of epidermal cell proliferation for the first stage of skin tumor promotion, the effect of hydroxyurea (HU), an inhibitor of DNA synthesis, on tumor formation was studied. Mice initiated with 7,12-dimethylbenz[a]anthracene received a single dose of phorbol 12-myristate 13-acetate (PMA) in stage I of promotion, followed by twice weekly application of the irritant skin mitogen phorbol 12-retinoate 13-acetate in stage II. A single dose of HU given intraperitoneally at different times before or after treatment with PMA was found to interfere with tumor formation, exhibiting an almost complete inhibition if administered 18 hr after PMA--i.e., at the time of maximal DNA synthesis. The inhibition of tumor formation by HU in the two-stage promotion experiment did not prevent a subsequent promotion of cells by repetitive PMA treatment. This indicates that the inhibitory effect of HU was due neither to cytotoxicity (killing of initiated cells) nor to an interference with initiation. The data indicate that epidermal DNA synthesis is obligatory for PMA-induced first-stage promotion. The causal relationship between both events remains to be established.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Cell Transformation, Neoplastic; DNA Replication; Female; Hydroxyurea; Mice; Mice, Inbred Strains; Phorbol Esters; Phorbols; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate

By means of a two-stage promotion protocol in mouse epidermis with 12-O-tetradecanoylphorbol-13-acetate as first-stage promoter and 12-O-retinoylphorbol-13-acetate as second-stage promoter, the effects of the former that are critical and obligatory for tumor promotion were shown to be irreversible in nature for at least 8 weeks. The reversibility of tumor promotion was related to the second stage of promotion, reflecting the reversibility of epidermal hyperplasia induced by 12-O-tetradecanoylphorbol-13-acetate.

Topics: Animals; Carcinogens; Cell Line; Epidermis; Female; Hyperplasia; Mice; Mice, Inbred Strains; Neoplasms, Experimental; Phorbol Esters; Phorbols; Precancerous Conditions; Skin Neoplasms; Tetradecanoylphorbol Acetate; Time Factors