Nutrition and carcinogenesis: historical highlights and future prospects.

Historical reviews were presented of several selected nutritional factors that are determinants of cancer incidence in laboratory experiments utilizing animals. An all-inclusive review of nutrition as it impacts cancer incidence was not done. Rather, the selection of subjects was based on a combination of several factors. (1) The efficacy of the factor as an inhibitor. (2) Current interest in the factor. (3) The extent to which the mechanisms of the inhibition is known and that knowledge may facilitate future studies. (4) The relevance to the human problem. The future of research on cancer prevention is bright. There are now mechanism-based rapid assays to detect food stuffs that prevent cancer and to assay for the active compounds therein. The list of inhibitors shown in Table 8 will continue to grow. The challenge is to achieve universal application to the human population of appropriate dietary practices that include foods that provide the protective factors shown in Table 8.

Model systems for defining initiation, promotion, and progression of skin neoplasms.

A number of items that must be considered in designing and choosing a suitable model for initiation and promotion testing have been described. Although these items may seem complex, tests for initiation and promotion are, in reality, quite simple and provide a rational approach to carcinogen testing. Several tests have been described here and Eastin, elsewhere in this book, describes the validation of a simple and highly recommended test. The processes involved in initiation and promotion are qualitatively different. The criteria for concern about possible human hazards as well as for regulation of initiators and promoters should be based on these qualitative differences. Realistic appraisal of the risk must be based on the level and nature of the potential hazard. In particular, it must be recognized that promoting action is reversible, that a threshold exists, and that promotion is readily inhibited. Therefore, animal tests that differentiate between potential initiators and promoters are essential to enable a logical assessment of human risk and the implementation of appropriate protective measures based on scientific facts.

The in vitro characterization of the inhibition of mouse brain protein kinase-C by retinoids and their receptors.

The mechanism of the in vitro inhibition of Ca2+-, phosphatidylserine-dependent protein kinase C (PK-C)2 by the purified holo (ligand-saturated) forms of cellular retinol-binding protein (cRBP) and cellular retinoic acid-binding protein (cRABP) was studied. We report here that the PK-C-inhibitory action of holo-cRBP and holo-cRABP is due to their respective ligands, all-trans-retinol and all-trans-retinoic acid; the reduced phosphorylation of the holo-retinoid-binding proteins and brain cytosolic proteins is not the result of a retinoid-induced soluble phosphatase or protease activity; retinoids reduce PK-C affinity for calcium and phosphatidylserine in vitro; and the structure-function activity of the retinoids and the specific interaction of these compounds with their binding proteins are important in blocking the activity of PK-C. These observations suggest that the inhibitory effect of retinoids on plasma membrane-associated PK-C activity pays a significant role in defining the early epigenetic aspects of PK-C-dependent tumor promotion and may be a physiological mechanism by which retinoids induce terminal differentiation in cell types that do not express soluble retinoid-binding proteins.

Absence of phosphorylation of retinoid-binding proteins by protein kinase C in vitro.

Cellular retinol-binding protein, cellular retinoic acid-binding protein, and fetal cellular retinol-binding protein were purified to homogeneity and each polypeptide had a molecular weight of 16,000. Their apoproteins were not phosphorylated under the same conditions. Their holoproteins did not inhibit the phosphorylation of histone III-S by protein kinase C. Each of these observations is contrary to the results reported by Cope et al. (Biochem. Biophys. Res. Commun., 120, 593-601, 1984).

The role of prostaglandin E1 in ornithine decarboxylase induction by tumor promoters.

The effect of topical application of PGE on induction of ODC in mouse epidermis was measured. When direct induction of ODC by TPA was blocked by also applying indomethacin, maximum ODC activity occurred only when PGE was applied simultaneously with TPA 4 1/2 hr before killing of the mice. If either TPA or PGE was applied at other times, ODC activity decreased substantially. Induction of ODC by mezerein was blocked by indomethacin but restored by PGE, as was observed with TPA, but induction by ethyl phenylpropiolate was not affected by indomethacin or PGE. DMBA did not cause a consistent increase in ODC activity, nor was its inductive action affected by indomethacin or PGE. However, another weak inducer, acetic acid, exhibited elevated ODC activity when PGE was also applied. Inhibition by topical retinoic acid of ODC induction by TPA was partially overcome in a dose-response fashion by PGE. The results indicate that at least 2 events, elevation of PGE and another independent event, are required for induction of ODC activity. It appears that TPA causes at least 4 independent events essential for tumor promotion. A model for the events in the 2-stage tumor promotion model is proposed.

Tumor promoter-induced refractory state against ornithine decarboxylase induction by 12-O-tetradecanoylphorbol-13-acetate in mouse epidermis.

More than one application of the potent tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), to mouse skin at intervals of more than 48 h led to a larger induction of ornithine decarboxylase (EC 4.1.1.17; ODC) than did a single application. In contrast, at intervals of less than 24 h, the first application of TPA appeared to induce a refractory state; the second application of TPA did not induce ODC. The extent of the inhibitory effect caused by the first application of TPA was dependent on the dose. The abilities of a series of phorbol esters to induce the refractory state correlated with their promoting abilities. However, both mezerein and ethylphenylpropiolate, potent hyperplastic agents with little or no promoting properties, induced the refractory state. On the other hand, pretreatment with TPA caused a refractory effect on ODC induction by mezerein but potentiated ODC induction by ethylphenylpropiolate. The epidermal cells escaped from the refractory state by repeated application of TPA at intervals of 24 h as well as at intervals of twice a week; that is, there was a full induction of ODC activity following a second application within 24 h of a prior application. TPA did not elicit production of detectable ODC-antizyme activity in mouse epidermis. Mixing of a soluble extract from mouse epidermis in the refractory state with that from TPA-stimulated epidermis gave essentially additive ODC activity. Elimination of ODC induction by topical application of retinoic acid or injection of cycloheximide concurrent with the first application of TPA did not restore the ability of a second application of TPA to induce ODC. These results suggest that the refractory effect on ODC induction by TPA does not result from feedback regulation of ODC.

12-O-tetradecanoylphorbol-13-acetate promotes tumors prior to initiation in two-stage promotion.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the non-promoter mezerein both induce ornithine decarboxylase activity in mouse epidermis by a route which can be blocked by indomethacin. In two-stage tumor promotion experiments in mice with mezerein as the stage II promoter, TPA was effective as the stage I promoter whether it was applied before or after an initiating dose of 7,12-dimethylbenz[a]anthracene (DMBA). There appear to be at least 4 events in promotion, only 3 of which are caused by second stage promoters.

In vitro induction of human skin ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

A method was developed for the in vitro induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) of ornithine decarboxylase (ODC) activity in human skin punch biopsy samples. Addition of TPA to 1 ml serum-free minimum essential medium containing a single 3-mm human skin punch biopsy sample obtained from a surgical specimen resulted in an induction of ODC activity with a peak activity at 5 hours after TPA addition. In vitro induction of human epidermal ODC activity was dependent on the TPA concentration in the medium; about a twofold increase in ODC activity was observed 6 hours after the addition of 0.1 microM TPA, and about a fivefold increase in ODC activity was observed with 1 microM TPA. TPA also caused about a fivefold to sixfold increase in ODC activity in 3-mm skin punch biopsy samples from healthy volunteers. Human skin punch biopsy samples remained responsive to TPA induction of ODC activity even when stored in serum-free medium at 4 degrees C for 24 hours. A similar degree of induction of ODC activity by TPA was observed whether whole unfractionated human epidermis or a soluble epidermal extract was used for ODC assays. Increased ODC activity was the result of the increase in enzymatically active ODC protein, quantitated by a [3H]difluoromethylornithine-binding assay. Thus human skin, like mouse skin, is responsive to TPA for ODC induction.

Inhibition of phorbol ester--induced human epidermal ornithine decarboxylase activity by oral compounds: a possible role in human chemoprevention studies.

Extensive animal data have suggested that, in some systems, the induction of ornithine decarboxylase (ODC) is an essential, although not sufficient, aspect of tumor promotion and that compounds that inhibit ODC can inhibit tumor formation. Using fasting human volunteers, we report that human epidermal and dermal ODC are consistently induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in a manner similar to that seen in mouse skin. There is a marked intersubject variation in TPA-induced epidermal ODC activity levels. Orally administered compounds significantly inhibited TPA-caused human epidermal ODC induction. These data may be useful in the further development of drugs, doses, and dose schedules for use in human cancer chemoprevention studies.

Induction of mouse brain ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate is independent of TPA receptor concentration.

Ornithine decarboxylase (ODC, E.C. 4.1.1.17) activity was measured in a 35,000 X g brain supernatant fraction, prepared 5 h after intracisternal injection of 12-O-tetradecanoylphorbol-13-acetate (TPA) into developing mouse brain. TPA-dependent induction of ODC activity was maximal on days 5 and 9 postnatally while on day 7, the developmental (endogenous) level of ODC in brain was high and, concurrently, the ability of TPA to induce ODC was reduced. Both TPA-dependent and developmental increases in mouse brain ODC activity were significantly reduced by intracisternal injection of retinoic acid (RA). The efficacy of TPA in elevating ODC activity at postnatal ages 1-220 days-old was independent of both soluble-and particulate-associated TPA receptor concentration. These observations suggest that although TPA receptor activation may be an obligatory event in ODC induction, TPA receptor activation and its concentration per se, are not sufficient determinants for ODC induction and tumorigenesis. Furthermore, the endogenous mechanism of ODC induction is distinct from that of the TPA-dependent increase in ODC enzyme activity.

Retinoid-binding proteins are phosphorylated in vitro by soluble Ca+2- and phosphatidylserine-dependent protein kinase from mouse brain.

Cellular retinol-binding protein (cRBP) and cellular retinoic acid-binding protein (cRABP) were purified from calf liver and uterus, respectively. Soluble Ca+2- phosphatidylserine-dependent protein kinase-C (PK-C) derived from mouse brain was capable of phosphorylating both of these apoproteins in vitro as determined by the phosphocellulose binding assay. The Km value was determined to be 6.2 microM for apo-cRBP and 5.1 microM for apo-cRABP. In contrast, the Km value for the histone III-S fraction was estimated to be 10.8 microM; the Km values for ATP in the presence of apo-cRBP and apo-cRABP were 12.4 microM and 2.6 microM, respectively. Specificity of phosphorylation of the retinoid-binding proteins was confirmed by polyacrylamide gel electrophoresis and subsequent autoradiography of the assay mixture as well as by a concentration-dependent, Ca+2, and phosphatidylserine sensitivity of the phosphorylation of both apo-cRBP and apo-cRABP. Inhibition of PK-C activity by holo-cRBP and holo-cRABP was also observed. Thus, phosphorylation of both of the retinoid-binding proteins may play an important modulating role in i) the ability of retinoids to function as antipromoters in chemically-induced tumorigenesis and ii) the control of physiological aspects of retinoid action in normal and retrodifferentiated cells.