12-o-retinoylphorbol-13-acetate and Cell-Transformation--Neoplastic

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

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

The biological activities in vitro of the incomplete (second-stage) tumor promoter, 12-O-retinoyl phorbol-13-acetate (RPA), and the complete tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) were compared. The doses of TPA and RPA necessary to inhibit the specific binding of [3H]-phorbol-12,13-dibutyrate ([3H]PDBu) to BALB/c 3T3 cells (50% inhibition doses; ID50; 8-13 ng/ml) were very similar; however, RPA was less potent than TPA in inhibiting [3H]-PDBu binding to Friend erythroleukemia cells (FELC). Intercellular communication between BALB/c 3T3 cells, measured by transfer of microinjected fluorescent dye (Lucifer Yellow), was inhibited by RPA as well as by TPA; TPA was about five times more potent than RPA. RPA also inhibited FELC differentiation induced by hexamethylene bisacetamide (HMBA) but not the differentiation of a TPA-resistant clone. The dose-responses of these two compounds in inhibiting differentiation of both TPA-sensitive and resistant FELC were very similar. When TPA and RPA were compared in their promoting activity of in vitro cell transformation of BALB/c 3T3 cells initiated with 3-methylcholanthrene (MCA, 0.1 microgram/ml), both TPA and RPA significantly increased the yield of morphologically transformed foci, and RPA was approximately 10 times more potent than TPA. These results suggest that RPA and TPA share many common in vitro biological effects and that these in vitro studies do not allow us to delineate clearly the effect of a second-stage tumor promoter from that of complete tumor promoters such as TPA.

Topics: Animals; Cell Communication; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Mice; Mice, Inbred BALB C; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; 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

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

Stimulation of epidermal growth in adult mouse skin can be induced by chemical agents, such as phorbol esters and other skin mitogens, or by mechanical means, such as skin massage and skin wounding. It leads to different kinds of epidermal hyperproliferation, according to interference with mechanisms of endogenous growth control (G1 chalone) and to mediation by endogenous regulatory factors (prostaglandins). Certain phorbol esters and skin wounding induce epidermal hyperproliferation and, in addition, a metaplastic process. Another property of these metaplasiogenic mitogens is their tumor-promoting efficacy in mouse skin, which has been initiated by a carcinogen in a subthreshold dose. Tailor-made phorbol esters allow the subdivision of the process of tumor promotion into two stages. In the first--probably irreversible--stage, a single application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or wounding brings about the events critical and obligatory for promotion, whereas in the second--probably reversible--stage, repetitive applications of an "incomplete promoter" evoke epidermal hyperplasia necessary to make the tumors visible. Adult guinea pig epidermis in vivo, as well as primary cell cultures derived from adult guinea pig ear epidermis, responds to the proliferative effects of phorbol esters such as TPA along a similar sequence of biochemical events as mouse skin in vivo. The in vitro approach allows the study of the molecular events involved in the mechanism of action of phorbol esters in more detail.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Mice; Phorbol Esters; Phorbols; Prostaglandins; Skin; Tetradecanoylphorbol Acetate