A B S T R A C T Natural killer cells spontaneously lyse certain tumor cells and may defend against malignancy. We have previously shown that natural killing (NK) by human peripheral blood mononuclear cells (PBMC) is suppressed in vitro by phorbol diester tumor promoters, including 12-0-tetradecanoylphorbol-13-acetate (TPA). We here demonstrate that suppression of NK is mediated by monocytes or polymorphonuclear leukocytes (PMN) and that suppression is dependent on the generation of reactive forms of molecular oxygen (RO), particularly hydrogen peroxide (H202). NK was suppressed not only by TPA but also by opsonized zymosan (yeast cell walls), which, like TPA, was not toxic to PBMC. Both TPA and zymosan stimulated the production of superoxide anion (O°) and H202 by PBMC. Production of RO correlated with suppression of NK. When PBMC were depleted of monocytes, the production of RO and the suppression of NK were both markedly reduced. Suppression could be restored by monocytes or PMN, both of which produced RO Suppression of NK by TPA was inhibited by catalase. Bovine superoxide dismutase had a limited effect on suppression, even in high concentration, and tyrosinecopper (II) complex, which also enhances dismutation of°2 to H202, had almost no effect on suppression.When H202 was directly generated enzymatically from glucose oxidase and glucose, NK was suppressed and suppression was reversed by catalase. NK was also suppressed by the enzymatic generation of°2 from xanthine oxidase and xanthine, but suppression under these conditions was again inhibited by catalase and not by superoxide dismutase, indicating that suppression was due to the secondary formation of H202 from O°. These results indicate that H202 is important in suppression of NK. Myeloperoxidase did not appear to play a role in suppression because inhibition of this enzyme by sodium azide, cyanide, or aminotriazole did not prevent suppression of NK. Suppression of NK was reversible; after exposure to zymosan, NK could be partially restored by the addition of catalase and superoxide dismutase or by the removal of zymosan. These studies demonstrate cellular regulation of NK by monocytes or polymorphonuclear leukocytes and indicate a role for RO in immunoregulation. INTRODUCTION Natural killer cells are mononuclear cells that spon-

The unusual case of a 12-year-old boy with well differentiated in situ, and focally invasive squamous carcinoma of the larynx is reported, together with a review of the medical literature. In addition to this one, 54 cases of laryngeal carcinoma in children 15 years of age or younger have been reported since 1868. All were squamous except for one adenocarcinoma of a laryngeal minor salivary gland. Carcinoma of the larynx in children parallels that of adults in terms of squamous histology, predominantly vocal cord involvement, pattern of local spread, and response to treatment. However, the incidence of female patients is higher in childhood (40% of childhood cases vs. less than 10% of adult cases), and risk factors, except previous irradiation of papillomas, are rare. Although unusual, carcinoma of the larynx in children is probably more common than thought and the diagnosis should be considered in any case of a child with persistent hoarseness or cough.

Evidence has been obtained that implicates the generation of reactive oxygen species as an early and critical event in the promotion of neoplastic transformation in mouse JB6 cells. The time courses for specific inhibition by CuZn-superoxide dismutase (CuZn-SOD) of the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced promotion of neoplastic transformation in JB6 cells and for changes in antioxidant enzyme activities associated with TPA-exposure were examined. The antipromoting effect of CuZn-SOD was found to be critically dependent on the time of addition of CuZn-SOD relative to the start of a 14-day exposure of cells to TPA. Treatment of JB6 P+ Clone 22 and Clone 41 cells with CuZn-SOD for 18 h before, simultaneously with or up to 1 h after exposure to TPA, all inhibited promotion of transformation maximally. Delay of addition of CuZn-SOD by 2 h or more after the start of TPA treatment resulted in a marked decrease in the promotion inhibitory effect. CuZn-SOD added 24 or 48 h after TPA had no effect on promotion of transformation. Exposure of JB6 cells to 0.2- (superoxide anion radical) generated exogenously by the aerobic xanthine oxidase reaction resulted in promotion of neoplastic transformation that was prevented by concurrent addition of CuZn-SOD. Taken together these studies provide evidence that increased superoxide anion generation within the first 2 h following TPA exposure is an essential event in promotion of transformation in JB6 cells. Upon TPA exposure, JB6 Clone 41 cells exhibited time-specific activity changes in the cellular SOD, glutathione peroxidase (GSH-Px), and catalase. SOD and GSH-Px activities were reduced to 54% and 26% respectively of basal levels within 2 h of TPA treatment. GSH-Px activity recovered to basal levels within 4 h and CuZn-SOD within 48 h. Catalase activity was maximally reduced to 50% of basal within 1 h after TPA treatment and rebounded to greater than basal levels within 4 h. It is postulated that a c-kinase-dependent event induces rapid elevation of superoxide anion following TPA exposure and that this leads to reduced activity of antioxidant enzymes. Since antipromotion by exogenous CuZn-SOD is effective only during the first 2 h following TPA exposure, this suggests that the promotion-relevant 0.2- elevation is transient.

The role of reactive oxygen (RO) in the promotion of neoplastic transformation of JB6 mouse epidermal cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated using inhibitors of RO itself or RO generating systems of seven different types. Bovine erythrocyte CuZn superoxide dismutase (SOD) maximally decreased anchorage-independent (AI) colony induction by TPA in semi-solid agar in a dose-dependent manner to 10% of TPA control level. The inhibitory effect was specifically on induction of transformation, not expression of transformation. Copper (II) (3,5-diisopropylsalicylic acid)2, which exhibits biomimetic SOD activity, was also effective. Two enzyme eliminators of H2O2, catalase and glutathione peroxidase, failed to prevent TPA-promotion. Among three hydroxyl radical scavengers, D-mannitol and Na-benzoate were moderately active but tetramethylurea did not specifically inhibit AI colony induction by TPA. A quencher of singlet oxygen, 1,4-diazobicyclo-[2,2,2]octane was also inactive. Antioxidants blocked AI transformation by TPA moderately (n-propyl gallate and tannic acid) or weakly (BHA). BHT did not specifically inhibit promotion of transformation. The effects of three inhibitors of the arachidonic acid cascade were examined. NDGA and quercetin (lipoxygenase inhibitors) were moderately active but indomethacin (cyclooxygenase inhibitor) was much less active. Based on these results, we suggest that superoxide anion (O2-.) is required for promotion of transformation by TPA. H2O2 and 1O2 appear not to be required. Hydroxyl radicals and lipid peroxides, possibly associated with O2-. action or formed in the course of oxidative metabolism of arachidonic acid also appear to be required but to a lesser extent. Products of the lipoxygenase pathway of arachidonic acid metabolism but not the cycloxygenase pathway may be important in promotion of transformation by TPA in JB6 mouse epidermal cells. The epidermal cells themselves can be both the source of and the target of the reactive oxygen in promotion.

Treatment of mice with sustained high levels of P-estradiol leads to a reduction in natural killer cell activity and genetic resistance to bone marrow transplantation. The loss of natural killing does not seem to result from either humoral or immune suppression. Natural killer cells are thought to depend on the bone marrow, and it is notable that estrogens reduce natural killing at approximately the same time that they produce a loss of marrow due to osteoproliferation. Similarly, mice with congenital osteopetrosis are deficient in natural killing. However, changes in natural killing during and after treatment with estrogen do not correspond directly to changes in marrow volume. Estrogens are known to exacerbate spontaneous autoimmunity in NZB/NZW mice. The relationship between this effect and the effect of estrogen on natural killing is not clear. When natural killing is lowered in NZB/NZW mice by the in vivo administration of "Sr, autoimmunity is reduced.We have been studying the effects of P-estradiol on natural killer cells in the mouse (1,2). Such cells are stimulated by viral infection (3), and though they do not

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Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P+) and nonpromotable (P-) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12-o-tetradecanoyl-phorbol-13-acetate (TPA). Cellular DNA isolated from one of two P+ lines, JB6 Cl 41 or JB6 Cl 22, was CaPO4 precipitated and used to transfect the P-cell line JB6 Cl 30. At 7 days posttransfection, the cells were suspended in agar with or without TPA at 1.6 x 10-8 M and assayed 10 days later for TPA-dependent colony formation. Whether there exists a genetic basis for susceptibility to tumor promoters is one of the important unanswered questions in carcinogenesis. Answering this question with in vivo initiation-promotion models (1, 3) has been difficult because tumor promotion is assumed to occur in a minor subpopulation consisting of carcinogeninitiated or post-initiated cells. Because such "promotable" subpopulations of carcinogen-exposed cells have not yet been identified in animals, one must study heterogeneous populations. For studying the genetics of tumor promoter sensitivity, clonal lines of initiated or post-initiated cells in culture can be useful. To be analogous to the mouse skin initiation-promotion model, initiated cells should have two essential features: (i) they must be stably nonneoplastic, and (ii) they must require exposure only to a tumor promoter, not to a complete carcinogen, to become irreversibly neoplastic. Postinitiated cells, i.e., those which have undergone early-stage promotion (10, 24) should differ from initiated cells by requiring a relatively short period of exposure to tumor promoter. The mouse JB6 epidermal cell model which we have developed satisfies the criteria indicated above for post-initiated cells.JB6 cells, originally established as a nonclonal line from untreated primary BALB/c mouse epidermal cultures, are nontumorigenic and anchorage dependent (7) and retain an epidermal specific cell surface antigen (2). Exposure of these cells to phorbot esters and other tumor promoters irreversibly promotes anchorage independence and tumorigenicity (4). Promotion of anchorage independence occurs when JB6 cells are exposed to second-stage (24, 25) but not first-stage tumor promoters and is inhibited by second-stage, not first-stage, promotion inhibitors (8). The JB6 model system thus appears to 1182 on May 9, 2018 by guest

A B S T R A C T Tumor-promoting phorbol diesters were shown to suppress natural killing in vitro by human peripheral blood mononuclear cells. The inhibitory effect of different phorbol diesters and their analogues correlated with their potency as tumor promoters, the most effective agent being 12-0-tetradecanoylphorbol-13-acetate (TPA). Both peripheral blood cells and targets specifically bound TPA, and natural killing could be inhibited by pretreatment of either cell population with TPA, though this was less effective than direct addition of TPA to the assay. Cells that had been pretreated with TPA released TPA and metabolites of TPA during subsequent incubation in fresh medium. This release of TPA was evidently responsible for the inhibition of natural killing by pretreated target cells; in experiments where labeled and unlabeled target cells were mixed, pretreatment of unlabeled targets with TPA inhibited killing of labeled targets. Suppression of natural killing by TPA was greatly reduced when adherent cells were removed from the peripheral blood cells, suggesting that monocytes mediate suppression. Inhibition of natural killing by TPA provides a model for examining the regulation of natural killing. Suppression of natural killing by phorbol diesters may contribute to their activity as tumor promoters.