To investigate the role of oncogene activation in the pathogenesis of malignant tumors, we have studied the tumorigenic and metastatic properties of NIH/3T3 secondary transfectants (designated A51) containing an activated c-Haras-) gene derived from the human T24 bladder carcinoma cell line and compared them Tumors are classically defined as benign or malignant (1). Benign tumors are noninvasive growths that do not spread to distant organs. Unless located in a functionally vital site (e.g., brain), they pose little threat to the patient and usually can be removed surgically. In contrast, malignant neoplasms are readily invasive, metastasize to organs throughout the body, and eventually kill their host (1). The biochemical events that distinguish malignant from benign neoplasms remain unidentified. Work in several laboratories has implicated oncogene activation in the expression of tumorigenicity but the role of oncogenes in the pathogenesis of malignant versus benign tumors has received little attention. The NIH/3T3 cell line, although immortalized in vitro, is reportedly nontumorigenic and is widely used as a recipient cell line for detecting transforming gene sequences isolated from tumorigenic cell lines or tissues (2-6). For example, upon transfection with the activated ras gene, NIH/3T3 cells form foci in tissue culture, exhibit anchorage independence, and, in those experiments in which in vivo studies have been conducted, produce tumors in nude mice (6-10). However, the behavior of the tumors formed by transfected NIH/3T3 cells has not been rigorously evaluated and it remains unclear whether oncogene activation is an event associated strictly with the pathogenesis of benign neoplasms or whether activation is also an essential feature for expression of metastatic properties. We report that transfection with the activated c-Ha-ras-l gene accelerates the tumorigenicity and enhances the metastatic potential of NIH/3T3 Tumorigenicity and Metastasis. Tumorigenicity and spontaneous metastatic potential were assayed by inoculating mice with different cell doses in the footpad (i.m.) or the supraclavicular region (s.c.). Tumor size was monitored at the supraclavicular site every 2-3 days by caliper measurement. For studies on experimental metastasis, different numbers of cells were injected into the tail vein of nude mice. At autopsy the major organs of all animals were examined both grossly and histologically for evidence of metastases. Single sections were prepared from each organ except the lung, in which case multiple sections were examined.Detection of Activated c-Ha-ras Oncogene and Human Alu Sequences. For preparation of DNA (12), cell monolayers established from primary tumors or metastatic foci were dispersed into phosphate-buffered saline (Pi/NaCl), pelleted, rinsed, resuspended in 10 mM Tris HCl, pH 8.0/0.35 M NaCl/1 mM EDTA, lysed in 0.5% NaDodSO4, and treated for 4-12 hr with Pronase (0.1 mg/ml) at 37°C. DNA was extracted with phenol, ethanol precipitated, and dissolved in 10 mM Tris-HCl, pH ...
Metastasis is a complex process whereby tumour cells from a primary neoplastic growth disseminate throughout the body and establish secondary tumour foci in distant organs. Biochemical traits associated with, or essential for, the expression of the metastatic phenotype have not yet been identified. In the course of examining stimulation of the B16 murine melanoma adenylate cyclase by melanocyte-stimulating hormone (MSH) and by the diterpene forskolin, we noted that tumour cell clones isolated from common parent cell populations differed widely in their responses to these agonists. We report here that the accumulation of cyclic AMP induced by MSH or forskolin shows a strong positive correlation with the ability of B16 melanoma clones to form pulmonary tumour colonies when injected intravenously (i.v.) into syngeneic mice ('experimental metastasis'). In parallel in vitro analyses of cyclic AMP metabolism and in vivo assays of experimental metastasis using replicate cell preparations, highly metastatic tumour cell clones consistently show greater than a 30-fold increase in cellular cyclic AMP when exposed to MSH or forskolin. By contrast, clones with limited metastatic abilities respond to the same agonists with only a two- to threefold increase in cellular cyclic AMP. These data suggest that cyclic AMP metabolism is linked with biochemical pathways that are responsible for the formation of experimental metastasis by the B16 melanoma.
The endogenous tumor-associated macrophage content and recruitment of labeled peritoneal exudate cells into experimental murine B16 melanoma metastases has been examined at different stages in the progressive growth of metastatic lesions. The recruitment of thioglycollate-elicited peritoneal exudate cells and peritoneal exudate cells activated in vitro with muramyl dipeptide was studied. Tumor-associated macrophages and labeled peritoneal exudate cells were identified in paraffin sections by specific histochemical staining and their density in individual metastases measured morphometrically. The density of tumor-associated macrophages and exogenously recruited peritoneal exudate cells was high in very small lesions but decreased rapidly as a function of enlargement of metastases, MD:An; where MD is macrophage density, A is the cross-sectional area of the lesion and n is a negative number. No significant difference was observed in the recruitment of activated and nonactivated peritoneal exudate cells. These results suggest that decreased recruitment of macrophages from the circulation may explain the decrease in the density of tumor-associated macrophages as metastases grow and indicate that macrophage activation is not accompanied by enhanced localization and/or uptake of macrophages into metastases.
The regulation of adenylate cyclase in murine melanoma tumor cell clones with different metastatic capacities has been studied in intact cells and isolated membrane preparations. Analysis of the responses of intact cells from a number of B16 melanoma clones revealed that treatment with melanocyte-stimulating hormone (MSH) or the diterpene, forskolin, produced significantly greater accumulation of intracellular cyclic adenosine 3',5' monophosphate (cAMP) in strongly metastatic clones than in weakly metastatic tumor cell clones. In contrast, in isolated membranes from the same panel of clones, the extent of activation by forskolin but not by MSH correlated with metastatic capacity. Sodium fluoride and 5'-guanyl-beta-gamma-imidodiphosphate [Gpp(NH)p] also stimulated adenylate cyclase in isolated membranes but the extent of activation did not correlate with the metastatic behavior of the donor cells. A combination of forskolin and Gpp(NH)p proved to be a sensitive prospective indicator for identifying differences in the metastatic capabilities of individual B16 melanoma clones. Adenylate cyclase in membrane preparations from strongly metastatic B16 clones displayed synergistic activation but stimulation of the enzyme from weakly metastatic clones was less than additive. To test the generality of these findings, similar investigations were performed on B16-BL6 melanoma cells, a highly invasive subline of the B16 melanoma, and the K1735, an ultraviolet-light-induced murine melanoma arising in a different mouse strain (C3H). Consistent with their high metastatic potential, clones derived from the B16-BL6 melanoma displayed elevated levels of hormonally-stimulated adenylate cyclase, thereby confirming, for this tumor system, a close association between hormonal responsiveness and metastatic capacity. In contrast, K1735 melanoma cell clones exhibited significant interclonal variation in adenylate cyclase activity and metastatic performance, but no consistent relationship between the two traits was detected. Differences in the regulation and/or the intrinsic catalytic capacity of adenylate cyclase may account, at least in part, for the variation in hormonal responsiveness observed among B16 clones with distinct metastatic properties and suggest that cAMP-dependent molecular processes may be required for the expression of B16 melanoma experimental metastatic potential.(ABSTRACT TRUNCATED AT 400 WORDS)
The ability of B16 melanoma clones to form tumor colonies in the lung after i.v. injection (experimental metastases) correlates positively with their capacity to respond to activators of cyclic adenosine 3-, 5-monophosphate (cAMP) metabolism (such as melanocyte-stimulating hormone and forskolin). To investigate whether this relationship is causal, the cAMP responses of 4 B16 melanoma clones of differing colonizing potential have been examined in freshly established stock cell cultures, in pulmonary colonies in vivo and in cultures established from these lesions. In all cases, the cAMP responsiveness of the excised colonies (and cultures derived from them) mimicked the responsiveness of the cultures from which they arose. B16 clones exhibiting low cAMP responsiveness gave rise to few experimental metastases all of which were poorly responsive to activators of cAMP metabolism. Similarly, clones with high cAMP responsiveness formed multiple lung colonies which displayed a marked sensitivity to agents that stimulated cAMP production. Parallel experiments on the spontaneous metastatic behavior of the same clones revealed that the cAMP responsiveness of cells in the primary (intrafootpad) tumor and spontaneous metastatic lesions in the lung faithfully reflected the response profile of the original tumor-cell inoculum but no correlation was found between cAMP responsiveness and the capacity to form spontaneous metastases. These data suggest that cAMP-dependent events may influence the survival, arrest and organ colony formation by cells injected directly into the circulation but appear to be of little or no importance in determining the early event(s) involved in the evolution of spontaneous metastases prior to the entry of cells into the circulation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.