Up- and down-regulation of granulocyte/macrophage-colony stimulating factor activity in murine skin increase susceptibility to skin carcinogenesis by independent mechanisms.

The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in tumorigenesis is complex. On the one hand, GM-CSF can promote tumor cell growth, survival, and even metastasis. On the other hand, it can stimulate tumor cell rejection. In skin, it is early expressed after topic application of tumor-promoting agents and therefore may be responsible for changes that correlate with skin tumor promotion (e.g., epidermal hyperproliferation and inflammation). To analyze GM-CSF function in skin tumorigenesis, we generated transgenic mice epidermally overexpressing either GM-CSF or a GM-CSF antagonist. Both types of transgenic mice exhibited significantly increased numbers of benign tumors in a two-step skin carcinogenesis experiment using 7',12'-dimethylbenz[a]anthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-CSF displayed a significantly elevated carcinoma burden following a single-step carcinogenesis protocol consisting of tumor initiation only. Therefore, endogenous promotion is responsible for elevated tumor development in GM-CSF-overexpressing mice. In antagonist transgenic animals, an increased tumorigenicity of modified B16 tumor cells after cutaneous transplantation as compared with nontransgenic or GM-CSF transgenic mice was observed. Thus, the antitumor activity leading to the repression of tumor cell growth in control mice is GM-CSF dependent and is compromised in mice expressing the antagonist. We suggest that both, up-regulation and down-regulation of GM-CSF activity in skin, increase the incidence and growth of tumors via two independent mechanisms: endogenous tumor promotion in the case of increased GM-CSF activity and compromised tumor cell rejection in the case of decreased GM-CSF activity.

Epidermal overexpression of granulocyte-macrophage colony-stimulating factor induces both keratinocyte proliferation and apoptosis.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is released by keratinocytes in sizeable amounts only under pathological conditions, e.g., after topical application of a tumor promoter, in atopic dermatitis (AD), and after wounding. To study the biological function of this cytokine release, we generated transgenic mice that constitutively overexpress GM-CSF in the epidermis. An increase in the numbers of mast cells and Langerhans cells (LCs) in transgenics versus nontransgenic controls was observed but no severe inflammation. This is consistent with a central role of this cytokine in the development and maturation of LCs. Mitotic activity in the epidemnis of transgenic mice was elevated, but epidermal thickness and differentiation were normal. Homeostasis is maintained by an increase of apoptosis in the epidermis. We describe the differential expression of regulators of apoptosis and discuss a potential mechanism for this novel proapoptotic activity of GM-CSF on keratinocytes. Both stimulation of proliferation and promotion of apoptosis are of great relevance to tumorigenesis. The latter may be a means of removing damaged cells after genotoxic stress or injury.

Isolation and characterization of fragments of reduced and S-carbamidomethylated human growth hormone produced by plasmin digestion. I. Chemistry.

Reduced and S-carbamidomethylated human GH (RCAM-hGH) was digested with human plasmin, yielding a mixture of products. These were partially separated by chromatography on DEAE-cellulose, yielding three major fractions: Da and Db, which were equipotent with native hGH in the weight gain test; and Dc, which was about half as active as hGH. Each of these was further purified by gel filtration, yielding a number of subfractions which were characterized as follows: Da1 is a very stable noncovalent complex of residues 1--134 and 141--191 of RCAM-hGH; Da2 represents residues 20--41; Db1 is very similar to Da1, but appears to have lost one or more amide groups; Db3 represents residues 95--134; Dc2 is a heterogenous fraction containing a further deamidated version of Da1 and Db1 plus a similar complex of residues 42--134 and 141--191, apparently with some carboxyterminal heterogeneity; Dc3, like Db3, represents residues 95--134. The biological activities of these fragments are discussed in the accompanying paper. Earlier work has shown that native hGH, upon digestion with plasmin, is cleaved primarily at residues 134 and 140. It is shown here that when RCAM-hGH is digested with plasmin, in about 87% of the molecules at least one cleavage takes place in addition to those at residues 134 and 140.

Assay of pig growth hormone preparations for metabolic activities in the rat and in man.

The possible somatotropic effect in man of porcine growth hormone (pGH) and its plasmin digest has not been comprehensively studied before. For this purpose, pGH was digested with rat or human plasmin; acrylamide gel electrophoresis showed less than 10% native pGH remaining in the digests. Native pGH and the 2 types of plasmin digest possessed similar GH potency, as measured by the weight gain assay in the hypophysectomized rat: 1-2 IU/mg. In 7 GH-deficient children and 3 adults with myotonic dystrophy, we measured the capacity of human GH (hGH), pGH, and pGH plasmin digests to cause: a) the retention of N, P, K, Na, and Cl; b) a rise in plasma free fatty acids; c) a fall in plasma alpha-amino NL d) impaired glucose tolerance; and e) hyperinsulinemia. Human GH was active in all respects at minimal effective dosages of .0168 to 0.168 IU/kg BW(3/4) per day. The pGH preparations had no detectable effect at 0.532 I.U./kg BW(3/4)/day. The data show that in man pGH and its plasmin digests possess less than 1/30, less than 1/10, and less than 1/3 the anabolic, adipokinetic, and diabetogenic potencies of hGH, respectively.

Biological properties of plasmin digests of S-carbamidomethylated human growth hormone.

Reduction and carbamidomethylation of the intrachain disulfide bridges of human growth hormone did not destroy its ability to stimulate weight gain or cartilage metabolism in hypophysectomized rats. The reduced and alkylated hormone also stimulated glucose oxidation in isolated adipose tissue of hypophysectomized rats when added in vitro. When the S-carbamidomethylated hormone was incubated overnight with human plasmin, approximately 95% of the starting material was completely digested, as judged by polyacrylamide gel electrophoresis. The plasmin digest retained the ability to stimulate weight gain, cartilage metabolism, and glucose oxidation. A fraction consisting of two major electrophoretic components was isolated from the digest by chromatography on tsephadex G-50. This fraction possessed the biological properties of the whole digest.

Isolation and biological characterization of fragments of human growth hormone produced by digestion with plasmin.

The biologically active component of plasmin digested human growth hormone consisted of residues 1-134 attached to residues 141-191 by the disulfide bond between residues 53 and 165. This large fragment of the hormone retained the in vivo capacity to stimulate weight gain and cartilage metabolism in hypophysectomized rats and exhibited the diabetogenic property of the native hormone in partially pancreatectomized, dexamethasone-treated rats. This fragment also retained the in vitro ability to stimulate protein synthesis and amino acid and sugar transport into isolated diaphragm muscle of hypophysectomized rats, Furthermore, a smaller peptide (residues 1-134) derived from the large fragment by reduction and carbamidomethylation of the disulfide bond retained high activity in in vitro systems and the in vivo capacity to stimulate cartilage metabolism provided the peptide was injected intravenously. Thus the present studies demonstrate that many of the in vivo and in vitro metabolic effects of human growth hormone on tissues of hypophysectomized rats can be elicited with a peptide comprising the aminoterminal 134 amino acid residues of native human growth hormone.

Metabolic effects of plasmin digests of human growth hormone in the rat and man.

As a first step in our study of structure-function relationships among primate and non-primate growth hormones, human growth hormone (hGH) was subjected to the limited digestive activity of human plasmin. The lyophilized whole digest, containing less than 2% of unchanged hormone, had an average of 2.3 new amino-terminal groups per mole. The digest had the same potency as the native hormone (a) in causing weight gain in hypophysectomized rats; (b) in stimulating somatomedin production in hypophysectomized rats; (c) in stimulating upake of [(3)H]leucine into isolated diaphragm of hypophysectomized rats; (d) in accelerating transport of [(14)C]alpha-aminoisobutyric acid into isolated diaphragm of hypophysectomized rats; (e) in stimulating uptake of [3-0-methyl-(14)C]glucose by isolated adipose tissue of hypophysectomized rats; (f) in accelerating conversion of [(14)C]glucose to (14)CO(2) by isolated epididymal adipose tissue of hypophysectomized rats. The digest also caused glucosuria in partially pancreatectomized rats treated with dexamethasone. These metabolic actions of plasmin-digested hGH in the array of animal tests were confirmed by comparable effects elicited in 11 human subjects (nine pituitary-deficient children and adolescents and two nondeficient adults). A single injection of the plasmin digest caused an increase in plasma free fatty acids and a fall in plasma amino acids. Seven daily injections caused positive balances of nitrogen, phosphorous, sodium, and potassium, gain in body weight, and in two of three subjects impairment of glucose tolerance. The potency of the plasmin digest in producing these metabolic effects in man was comparable to that of native hGH.Thus, 2-3 bonds in the hGH molecule can be cleaved by plasmin without impairing the hormone's growthpromoting, anabolic, diabetogenic, and adipokinetic actions for rat and man.