R.K. Boutwell

Identification of a calcium- and phospholipid-dependent phorbol ester binding activity in the soluble fraction of mouse tissues

A soluble cytosolic protein which specifically and with high affinity binds tumor promoting phorbol ester tumor promoters was found in mouse tissues Calcium and phosphatidyl serine had to be included in the binding assay to obtain maximal binding activity by the soluble receptor. Soluble binding activity levels paralleled the levels of membrane associated binding activity with the soluble activity accounting for 27% to 80% of the total. Except the calcium and phospholipid requirement, no difference was found between the soluble and membrane associated receptors, suggesting that they are the same protein which can move between the soluble cytosol and cell membranes.

Polyamine biosynthesis and skin tumor promotion: Inhibition of 12-0-tetradecanoylphorbol-13-acetate-promoted mouse skin tumor formation by the irreversible inhibitor of ornithine decarboxylase α-difluoromethylornithine

Abstract Application of 12-0-tetradecanoylphorbol-13-acetate (TPA) to mouse skin leads to the induction of ornithine decarboxylase (EC 4.1.1.17) and the accumulation of putrescine. The relevance of these TPA-induced changes to the mechanism of tumor promotion was determined using α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase. α-Difluoromethylornithine applied to the skin of mice or administered in drinking water in conjunction with applications of TPA to 7,12-dimethylbenz[a]anthracene-initiated mouse skin inhibited the formation of mouse skin papillomas by 50 and 90% respectively; TPA-induced ornithine decarboxylase activity and the accumulation of putrescine were almost completely inhibited.

Direct measurment of specific binding of highly lipophilic phorbol diester to mouse epidermal membranes using cold acetone

Abstract Using acetone cooled to −78°C to wash a mouse epidermal membrane preparation after incubation with [ 3 H]12-0-tetradecanoylphorbol-13-acetate, nonspecific binding of the ligand is reduced by 100 fold allowing specific binding to be easily quantitated. This technique may be useful in binding studies of other highly lipid soluble chemicals to membrane constituents.

Puromycin-induced glycogenolysis as an event independent from inhibited protein synthesis in mouse liver; effects of puromycin analogs☆

Abstract To determine if puromycin-induced glycogenolysis is dependent upon inhibition of protein synthesis, the effect of puromycin and two of its analogs was studied on these parameters. Loss of liver glycogen and inhibition of hepatic protein synthesis following puromycin are not parallel effects on a dose response basis. If administered in 4 equally divided injections over a 4-hour period, puromycin aminonucleoside and 6-dimethylaminopurine as well as puromycin cause glycogen depression, but do not inhibit protein synthesis to a degree equivalent to puromycin. At 1 hour after a single injection, the relative potency to decrease hepatic glycogen was 6-dimethylamino-purine > puromycin aminonucleoside > puromycin, while the comparative degree of inhibition of glycine-2-C 14 incorporation into protein was reversed, with 6-dimethylaminopurine showing little effect. Administration of sodium pentobarbital was found to block the glycogenolytic response to puromycin at 1 hour without arresting puromycin-induced inhibition of protein synthesis. These results suggest that puromycin and its analogs cause depression of liver glycogen independently of their effect on protein synthesis.

The depletion of liver glycogen in puromycin-treated animals

Abstract Mice and rats receiving puromycin dihydrochloride intraperitoneally (6.0 and 60 mg. per animal, respectively) in four equally divided doses over a 4-hr, period showed complete depletion of hepatic glycogen and inhibition of incorporation of glycine-C 14 into liver protein. Administration of a single dose of puromycin to mice was followed by rapid inhibition of synthesis of liver protein with concurrent loss of glycogen from the liver. Carbohydrate levels in liver, blood, and muscle were followed in mice up to 4 hr. after a single injection of puromycin. The decrease in liver glycogen was accompanied by an increase in blood sugar levels while muscle glycogen content was not appreciably affected during this period.

Intracellular calcium and skin tumor promotion: Calcium regulation of the induction of epidermal ornithine decarboxylase activity by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Abstract The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin explants maintained in a serum-free Eagles HeLa cell medium. Chelation of extracellular calcium by ethyleneglycol-bis-(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Extracellular magnesium could not replace calcium for ODC induction by TPA. Concurrent incubation of skin pieces with a calmodulin inhibitor trifluoperzine (TFP) inhibited ODC induction. Furthermore, inclusion in the medium of lanthanum, which has a higher affinity for calcium-binding sites than calcium and displaces surface-bound calcium, inhibited ODC induction by TPA.

Retinoid-binding proteins are phosphorylated invitro 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.

Enzymatic oxidation and ruduction of retinal by mouse epidermis

Abstract Evidence is presented for the ability of mouse epidermis to oxidize retinal by the action of a cytosolic enzyme with a pH optimum of about 8.5. The enzyme activity was slightly enhanced by the addition of either NAD or FAD. The reaction exhibited Michaelis-Menton kinetics with a km of 1.627 × 10−4M for retinal. Mouse epidermal cytosol was also capable of reducing retinal. This enzyme had an optimum pH of 6.0 and the activity of the undialyzed enzyme was enhanced 5- to 7-fold by the addition of reduced NAD or NADP. Possible roles of these enzymic activities in epidermis are discussed.

Characterization of arginase activity from mouse epidermis and its relation to ornithine decarboxylase induction by the tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate

Arginase, which catalyzes the cleavage of L-arginine to urea and ornithine, was detected in both soluble and particulate fractions of mouse epidermis. In a typical experiment, about 75 and 25% of the total arginase activity was associated with the soluble (100 000 X g supernatant) and the washed particulate fraction, respectively. Both soluble and particulate enzymes required the presence of divalent Mn2+ for activity. Arginase activity was increased by about 50% in the particulate fraction, but not in the soluble fraction, by preheating the fractions at either 50 or 55 degrees C in the presence of 15 mM MnCl2. Enzyme activity in both fractions, in the absence of 15 mM MnCl2, dropped precipitously during heating. A comparison of the nature of arginases in the soluble and particulate fractions revealed similar Km values (13 mM) and pH optima (9.5) and identical heat denaturation curves. Application of 10 nmol of 12-O-tetradecanoylphorbol-13-acetate to mouse skin did not increase arginase activity in either fraction over a period of 24 h. In contrast, there was a large increase in ornithine decarboxylase activity in the soluble fraction 4.5 h after treatment. Mouse epidermal ornithine decarboxylase activity was much less than arginase activity and was predominantly localized in the soluble fraction. These results indicate that the normal level of arginase activity is not a limiting factor for the stimulation of polyamine biosynthesis by TPA. High arginase activity in mouse epidermis may play a role in providing ornithine for polyamine biosynthesis and in the production of glutamate and proline as well as in the production of keratinous proteins.

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.