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Clonal isolates of mouse 3T3 cells and primary rat embryo cells, recovered nonselectively after infection by simian virus 40 (SV40), have been tested for tumorigenicity in the immune-deficient nude mice in order to determine the cellular growth properties in vitro specifically correlated with neoplastic growth in vivo. In addition, mouse 3T3 cells transformed by murine sarcoma virus (MuSV, Kirsten strain), and revertants isolated from cells fully transformed by either SV40 or MuSV were also studied. Results suggest that the single cellular property consistently associated with tumorigenicity in nude mice is the acquisition by virus-transformed cells of the ability to proliferate in vitro in the absence of anchorage. Other cellular parameters of virusinduced transformation, such as lack of sensitivity to high cell density and the capacity to grow in low serum concentration, are dissociable from cellular tumorigenicity. This conclusion is supported further by the demonstration that specific selection in vivo for tumorigenic cells from anchorage-dependent cells results in the isolation of anchorage-independent cells. Conversely, a single-step selection in vitro for anchorage-independent cells from nontumorigenic cells results in a simultaneous selection of highly tumorigenic subclones. Infection of susceptible animal cells in vitro by tumor viruses usually results in a spectrum of stable alterations in cellular growth properties, as well as in the appearance of virus-specific antigens in the transformed cells (1). In particular, division in populations of untransformed cells is inhibited by any of the following three environmental constraints: extensive cell-cell contact (2), reduction of serum concentration (3, 4), or deprivation of a solid substrate for cell anchorage (5, 6).Recent results have demonstrated that cellular responses to the experimental parameters which differentiate the normal cell from its transformed counterpart are not coordinately controlled (7,8). Each constraint is the source of a selective assay that yields a different class of transformed cell line. Nonselective transformations of 3T3 mouse cells and of primary rat embryo cells by simian virus 40 (SV40) yielded lines displaying many different transformed phenotypes. While some lines were fully insensitive to each of the three constraints, most transformed lines lost only one or two of these constraints and remained normal for the others. Negative selection of revertant cell lines from a fully transformed 3T3 cell also dissociated these three parameters of growth control (9, 10).These observations suggested to us that not all of the altered cellular growth properties commonly associated with Abbreviations: SV40, simian virus 40; MuSV, murine sarcoma virus, Kirsten strain; RE, rat embryo; ME, mouse embryo. t Present address:

The role of thymic functions in the development of insulin-dependent diabetes was investigated in athymic nude (nu/nu) mice and euthymic heterozygous (+/nu) littermates of BALB/c origin treated with streptozotocin. The injection of a single high dose of streptozotocin (200 mg/kg body weight) induced rapid and permanent hyperglycemia in both nu/nu and +/nu mice. In contrast, the injection of the same total dose divided into multiple "subdiabetogenic" doses (40 mg/kg per day for 5 consecutive days) caused the development of delayed but progressive hyperglycemia only in the thymuscompetent +/nu mice. Female mice of either genotype were significantly less susceptible to streptozotocin at both doses. Restoration of thymic immunity in nu/nu mice by thymus grafts also restored the susceptibility to the hyperglycemic effects of multiple low doses of streptozotocin. Moreover, splenic lymphocytes from +/nu mice previously made diabetic with the multiple low-dose injections of streptozotocin induced transient glucose intolerance in nu/nu mice. The ability of the diabetic spleen cells to transfer the diabetic state was abolished when the splenic lymphocytes were depleted of the T cells but not when they were depleted of B cells. These results provide direct proof that thymus-dependent functions play an obligatory etiologic role in the development of diabetes in mice treated with repeated subdiabetogenic doses of streptozotocii. These observations also add to the growing evidence that autoimmune amplification mechanisms may be critically involved in the etiology of juvenile-tnset diabetes mellitus in humans.There is increasing evidence that the development of the juvenile-type insulin-dependent diabetes mellitus is mediated by autoimmune processes. Pancreatic islets of children with newly diagnosed insulin-dependent diabetes frequently contain infiltrating mononuclear lymphocytes (1), and a large fraction of juvenile-onset diabetes patients have both circulating antibodies against the islet cells (2) and lymphocytes with cytolytic activity against human insulinoma cells in culture (3). In addition, epidemiologic studies in several countries have independently established that a number of alleles of the HLA gene complex, especially of the HLA-D locus, are associated with a significantly increased risk of this form of diabetes (reviewed in ref. 4).Evidence implicating autoimmune processes in the etiology of insulin-dependent diabetes comes also from experimental model systems utilizing laboratory rodents. In a series of experiments, Like and Rossini and their coworkers have shown that the induction of diabetes in mice and rats by the beta-cell toxin streptozotocin typically proceeds in two quite different pathways. When a single, sublethal, high dose of streptozotocin was administered to susceptible animals, rapid destruction of the islet beta cells ensued, followed almost immediately by a profound and permanent hyperglycemia (5). In contrast, the administration of the same total dose of streptozotocin, divided into m...

Fibronectin (FN ; also called large external transformation-sensitive [LETS] protein or cell-surface protein [CSP]) is a large cell-surface glycoprotein that is frequently observed to be either absent or greatly reduced on the surfaces of malignant cells grown in vitro . Because FN may be a useful molecular marker of cellular malignancy, we have carried out an extensive screening to test the specific association among the degree ofexpression of FN, anchorage-independent growth, and tumorigenicity in the athymic nude mouse . A variety of diploid cell strains and established cell lines were tested for the expression of surface FN by indirect immunofluorescence using rabbit antisera against human cold insoluble globulin, rodent plasma FN, or chicken cell-surface FN . Concomitantly, the cells were assayed for tumor formation in nude mice and for the ability to form colonies in methylcellulose . Tumorigenic cells often showed very low surface fluorescence, confirming earlier reports . However, many highly tumorigenic fibroblast lines from several species stained strongly with all three antisera . In contrast, the anchorage-independent phenotype was nearly always associated with tumorigenicity in -35 cell lines examined in this study . In another series of expriments, FNpositive but anchorage-independent cells were grown as tumors in nude mice and then reintroduced into culture . In five of the six tumor-derived cell lines, cellsurface FN was not significantly reduced ; one such cell line showed very little surface FN .Our data thus indicate that the loss of cell-surface FN is not a necessary step in the process of malignant transformation and that the growth of FN-positive cells as tumors does not require a prior selection in vivo for FN-negative subpopulations .KEY WORDS fibronectin . cell studied in vitro . These generally include decreased transformation . anchorage independencesensitivity to factors which restrict the growth of tumorigenicity . nude mouse normal cells, such as low serum concentration (8, 47), lack of a solid surface for anchorage (26, 52, Malignant transformation of animal cells involves 53), and contact inhibition (6,57,58) . In addition changes in many cellular properties which can be to their greater cellular growth autonomy in vitro, J . CELL BIOLOGY

Susceptibility of mice to experimental insulin-dependent diabetes as induced by multiple subdiabetogenic doses of streptozotocin has been shown to be strongly gender-dependent, males being much more susceptible than females. We examined this gender difference further in two strains of genetically susceptible mice to determine whether exogenous steroid sex hormones can both suppress the high susceptibility of males and potentiate the low susceptibility of females. Our results show that, in both BALB/cBOM and C57BL/6 mice, exogenous estrogens can suppress the high susceptibility of males. Conversely, the normally streptozotocin-resistant females become as highly susceptible as males after the administration of androgens. The inhibitory effect of estrogens and the potentiating effect of androgens can be demonstrated after the hormones are given to the mice either chronically (in slow-release capsules implanted at a subcutaneous site), or immediately prior to streptozotocin injection. These observations are consistent with the view that the critical factor that determines the susceptibility of mice to the hyperglycemic effects of streptozotocin is not the absolute concentration of androgens per se, but rather the relative overall level of androgens over estrogens in the recipient animal. Several alternative mechanisms for the effect of sex hormones on diabetogenic sensitivity are discussed.