Adenosine deaminase activity associated with phagocytic vacuoles. Cytochemical demonstration by electron microscopy.

Adenosine deaminase activity has been localized within the cell membrane and it surrounds phagocytic vacuoles in mouse macrophages. Adenosine deaminase is thus strategically located to direct metabolic flux through the enzymes of the purine catabolic pathway. Xanthine oxidase, a key enzyme of this pathway, produces superoxide during its reaction with its substrates. Enzyme activity was visualized for electron microscopy by means of hydrolysis of 6-Chloropurine ribonucleoside to produce Cl-, which is precipitated with Ag+. The latter is converted into Ag0 by light, and the resulting deposit is visualized with the electron microscope.

Immunomodulation of murine B16 melanoma metastasis: thymosin, thymectomy and irradiation.

Thymosin, a product of the endocrine system, was used to further define the effects of immunomodulation of metastasis. Adult thymectomized C57BL/6 mice, 4 wk post-irradiation (400 R) had a decrease in the number of pulmonary metastasis (compared to controls) following tail vein injection of 5 X 10(4) B16 melanoma cells. Thymosin fraction 5 (fr. 5) administration (200 micrograms/mouse, 3 times weekly beginning 2 days post-thymectomy) returned the number of metastasis to the nonthymectomized values. Thymosin treatment of sham-operated, sham-operated irradiated, or thymectomized nonirradiated mice did not significantly elevate the number of metastases compared to the respective controls. Variant tumors which have an increase in metastasis following thymectomy and irradiation were also used. Thymosin administration reversed the effects of thymectomy in such variants, resulting in a decrease in metastasis. Metastases in thymosin-treated control mice were not significantly altered. A role for the thymus in metastasis via an endocrine product (thymosin) is suggested by these studies. Since thymosin did not increase metastasis in intact mice with tumors, further clinical trials with thymosin in cancer patients are not counterindicated by our results. These experiments confirm that thymosin fr. 5 is an important probe of the immunoendocrine events involved in tumor growth and metastasis.

Effects of 1,2-symmetrical dimethylhydrazine on jejunocolic transposition in Sprague-Dawley rats.

In this experiment, a segment of the left colon including the upper part of the rectum was transposed to the upper jejunum, and a segment of upper jejunum was transposed to the left colon of the same animal. In another group, the same colon and jejunum segments were transsected and reanastomosed in place. A third group served as a normal control. After a recovery period, weekly s.c. 1,2-symmetrical dimethylhydrazine injections were begun. Each animal received a total of 20 injections at a dose of 20 mg/kg. Five weeks after the last 1,2-symmetrical dimethylhydrazine injection, 15 of 19 (79%) of the animals had one or more tumor(s) in the transposed colon segment, while none had tumor in the transposed jejunal segment. Transsected and reanastomosed animals showed the same distribution of tumors as did the normal control animals. All three groups had tumors at other sites in the colon and rectum. In addition, about 20% had tumors of the duodenojejunal area. These data indicate that the colonic mucosa is the primary target for the carcinogenic effect of 1,2-symmetrical dimethylhydrazine, independent of other variables such as the fecal stream.

Glucocorticoid receptors and the effect of glucocorticoids on the growth of B16 melanoma.

The glucocorticoid receptors were measured and characterized in the transplantable B16 murine melanoma using [3H]-dexamethasone by a charcoal adsorption technique. In the tumor cytosols assayed, the levels of receptors ranged from 44 to 200 fmol/mg protein, and the corresponding Kd's ranged from 2 to 43 nM. Sucrose density gradient analysis showed a peak sedimenting at 7.1S under low-ionic-strength buffer which was completely eliminated with a 100-fold molar excess of unlabeled triamcinolone acetonide in the incubation mixture. This peak of bound radioactivity shifted to the 4.4S region under high-ionic-strength buffer (0.4 M KCl) conditions. Competition experiments, using [3H]dexamethasone and various unlabeled steroids at a 100-fold molar excess, showed characteristics typical of glucocorticoid receptors seen in other tissues. Administration of various glucocorticoids, e.g., dexamethasone, hydrocortisone acetate, and prednisolone, in different doses and regimens showed a marked and significant inhibition of tumor growth as measured by mean tumor diameter and weight. Although glucocorticoid treatment does not seem to affect the incidence of pulmonary metastases, the number of pulmonary nodules appears to be significantly greater in some groups treated with higher doses of these drugs. In survival experiments, administration of hydrocortisone acetate in various doses and regimens also resulted in a significant increase in the median survival of mice compared to 0.9% NaCl solution-treated controls. These results indicate that the growth inhibition of B16 melanoma by glucocorticoids may be a direct effect mediated by interaction with the glucocorticoid receptor.

Correlations between the leukocyte adherence inhibition microassay and in vivo tests of transplantation resistance.

The leukocyte adherence inhibition (LAI) microassay detects tumor-associated antigen(s). Extracts of colon carcinoma (MCA-38 and B16 melanoma tumors, both syngeneic to the C57BL/6J mice) are recognized only by peritoneal cells from mice bearing the corresponding tumor. To ascertain whether this in vitro antigenic recognition correlates with the ability of the host to recognize and reject a tumor in vivo, serial LAI microassays were performed synchronously with experiments designed to test the ability of mice bearing tumors to reject live secondary tumor challenges. Concomitant tumor immunity was present in the MCA-38 tumor-bearing mice on 3 occasions from 5 to 15 days from primary inoculation. In the B16 system, concomitant immunity was present on one occasion 10 days after primary inoculation. These results in turn were paralleled with the specific in vitro recognition of tumor antigens as detected by the LAI microassays. Loss of immunity in the "eclipse" phase of tumor development, as detected by concomitant tumor immunity, was paralleled by nonreactivity of the indicator cells in the LAI microassay.

Genetics of colon carcinogenesis in mice treated with 1,2-dimethylhydrazine.

Genetic analysis of colon tumor induction by symmetrical 1,2-dimethylhydrazine (DMH) was undertaken in F1, F2, and reciprocal backcross hybrids derived from a cross between two inbred mouse strains, the 100% susceptible ICR/Ha and completely resistant C57BL/Ha. Mice, 12 to 14 weeks old, received 22 successive weekly s.c. injections of 0.35% aqueous solution of DMH buffered to pH 6.5. A dose of 15 mg/kg/mouse/week produced invasive colon adenocarcinomas in all ICR/Ha males and females (60 of 60) within 22 weeks. None of the 90 C57BL/Ha mice developed DMH tumors during 44 weeks of observation. Susceptibility to the carcinogen was dominant, as indicated by 100% colon tumor incidence in reciprocal ICR/Ha X C57BL/HaF1 hybrids (68 of 68) and in the susceptible backcross ICR/Ha X F1 (42 of 42). Tumor yield in F2 hybrids (94 of 120) was 78%, which is in close agreement with the 3:1 ratio expected if a single dominant DMH susceptibility gene is inherited via the F1 from the ICR/Ha grandparent. Likewise, tumor yield in resistant backcross mice of genotype C57BL/Ha X F1 (46 of 117) is not out of line with the anticipated 1:1 ratio in the latter type of test hybrids. Tests with five isozyme markers and two coat color genes have tentatively ruled out linkage of DMH susceptibility on seven autosomes. The 47% tumor incidence among 57 male resistant backcross hybrids, regardless of whether their single X chromosome was inherited from the ICR/Ha or C57BL/Ha strain, provides evidence against sex linkage.