TGF-beta 1 and skin carcinogenesis: antiproliferative effect in vitro and TGF-beta 1 mRNA expression during epidermal hyperproliferation and multistage tumorigenesis.

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.

Epidermal G1-chalone and transforming growth factor-beta are two different endogenous inhibitors of epidermal cell proliferation.

Epidermal G1-chalone and transforming growth factor-beta (TGF beta), two endogenous inhibitors of epidermal cell proliferation, were compared with regard to several effects on epidermis in vivo and in vitro. Both factors inhibited DNA labeling in a rat tongue epithelial cell line, with similar kinetics and half-maximal effects at approximately 1 pg/ml (enriched chalone) and 1 ng/ml (TGF beta). For primary neonatal mouse keratinocytes, TGF beta was found to be a rather strong inhibitor of cell proliferation, whereas chalone showed only a weak effect on cells grown in medium containing 1.2 mM Ca2+ and no effect at all in the presence of 0.06 mM Ca2+. Vice versa, upon i.p. injection, only chalone was able to inhibit mouse epidermal DNA synthesis in vivo, whereas TGF beta had no effect at all. A moderate increase of transglutaminase activity in neonatal primary mouse keratinocytes was induced by both factors at concentrations of about 300 pg TGF beta/ml and 10 pg chalone fraction/ml. Chalone did not compete with [125I]TGF beta for specific binding sites on primary murine keratinocytes. A polyclonal "chalone antiserum" did not interact with TGF beta, and a neutralizing TGF beta antibody that inhibited the effect of TGF beta on cell proliferation could not block the inhibitory effect of chalone on RTE2 cells. In contrast to TGF beta, epidermal G1-chalone did not induce proliferation of NIH-3T3 cells. These results indicate that epidermal G1-chalone and TGF beta are two different inhibitors of epidermal cell proliferation.

Stimulatory role of transforming growth factors in multistage skin carcinogenesis: possible explanation for the tumor-inducing effect of wounding in initiated NMRI mouse skin.

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.

The epidermal G1-chalone: an endogenous tissue-specific inhibitor of epidermal cell proliferation.

An apparently macromolecular factor is isolated from aqueous skin extracts which inhibits DNA synthesis in vivo and in vitro with high efficacy (ID50 in vivo 0.2 pmol/g, in vitro 0.2 pM) and in a highly specific manner showing a point of attack in the late G1-phase of the cell cycle (epidermal G1-chalone). Preliminary characterization indicates an unusual highly amphipathic structure consisting of amino acids and carbohydrate. Despite its apparent molecular weight of approximately 10 kD the chalone is stable against denaturing agents and most enzymes, including proteases. An inverse correlation between chalone responsiveness of mouse epidermis in vivo and the development of hyperplasia due to injury indicates an important role of the factor in the regulation of tissue homeostasis. According to its physicochemical and biological properties the epidermal G1-chalone appears not to be related to other endogenous inhibitors of epidermal cell proliferation such as the pentapeptide pyroGlu-Glu-Asp-Ser-GlyOH and transforming growth factor beta (TGF beta).